The current pandemic of sexually transmitted human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) has created an urgent need for a new type of microbicide, one that is both a spermicide and a virucide. In a systematic effort to identify a non-detergent-type antiviral spermicide, we have rationally designed and synthesized a series of cyclohexenyl thiourea (CHET) nonnucleoside inhibitors (NNIs) of HIV-1 reverse transcriptase (RT) with sperm-immobilizing activity (SIA). To gain further insight into the structural requirements for the optimal activity of these dual-function NNIs, we compared the effects of thiazolyl, benzothiazolyl, and pyridyl ring substitutions and functionalization with electron-donating and electron-withdrawing groups as well as the importance of thiourea and urea moieties of 15 heterocyclic ring-substituted NNIs. RT activity and p24 antigen production in HIV-infected peripheral blood mononuclear cells were used as markers of viral replication. Computer-assisted sperm analysis was used for evaluating SIA of CHET compounds. The rabbit model was used for evaluation of in vivo mucosal toxicity and contraceptive activity of the lead NNIs. Three CHET-NNIs with a bromo, chloro, or methyl substitution at the 5 position of the pyridyl ring exhibited potent anti-HIV activity at nanomolar concentrations (IC₅₀ = 3–5 nM) and SIA at micromolar concentrations (EC₅₀ = 45–96 μM). The dual-function CHET-NNIs were potent inhibitors of drug-resistant HIV-1 strains with genotypic and phenotypic NNI resistance. Upon substitution of the sulfur atom of the thiourea moiety with an oxygen atom, the most striking difference noted was a 38-fold reduction in time required for 50% sperm immobilization (T_1/2). A quantitative structure-activity relationship (QSAR) analysis was used in deriving regression equations between 20 physicochemical properties and SIA of NNIs. QSAR analysis showed that the T_1/2 values positively correlated with values for molecular refractivity (r = 0.88), hydrophobicity (r = 0.72), atomic polarizability (r = 0.70), and principal moment of inertia (r = 0.63) of spermicidal NNIs. A stepwise multiple regression model to describe the relationship of T_1/2 values with these four regressors provided excellent predictability (r = 0.93). Exposure of semen to thiourea/urea NNIs either alone or in combination at the time of artificial insemination led to marked or complete inhibition of pregnancy in rabbits as assessed by the number of embryo implants versus corpora lutea on Day 8 of pregnancy. Repeated intravaginal application of a gel-microemulsion with and without 0.5%, 1%, and 2% CHET-NNI or its urea analog either alone or in combination did not induce mucosal toxicity. We hypothesize that the gain of spermicidal function by CHET-NNIs is due to their metabolic oxidation to urea analogs by sperm. Three reaction pathways are discussed. The extremely rapid SIA of the urea analog as well as the broad-spectrum anti-HIV activity of spermicidal CHET-NNIs together with their lack of mucosal toxicity and the marked ability to reduce in vivo fertility is particularly useful for the clinical development of a dual-function spermicidal microbicide. The cyclohexenyl pyridyl NNIs, especially N-[2-(1-cyclohexenyl)ethyl] N′-[2-(5-bromopyridyl)]-thiourea in combination with the urea analog, show unique clinical potential as anti-HIV spermicides aimed at curbing the sexual transmission of multidrug-resistant HIV-1 while providing effective fertility control for women.