We previously demonstrated that bis-cyclopentadienyl (Cp) complexes of vanadium(IV) (vanadocenes) are potent spermicidal and apoptosis-inducing agents. To gain further insight into the structure-function relationships controlling these two properties of vanadocenes, we have synthesized analogues in which the bis-Cp rings were substituted with one or five electron-donating methyl groups. The three complexes included vanadocene dichloride (VDC), bis(methylcyclopentadienyl) vanadium dichloride (VMDC), and bis(pentamethylcyclopentadienyl) vanadium dichloride (VPMDC). The concentration-dependent effect of these vanadocenes on sperm-immobilizing activity (SIA), mitochondrial membrane potential (ΔΨm), axonemal dynein ATPase activity, and tyrosine phosphorylation of global and axoneme-specific sperm proteins was assessed by computer-assisted sperm analysis, flow cytometry, colorimetry, and immunoblotting, respectively. Apoptosis-inducing ability was quantitated by the two-color flow cytometric terminal dideoxynucleotidyl transferase-based assay that labels 3′-hydroxyl ends of fragmented DNA. All three vanadocenes induced rapid sperm immobilization (T_1/2 < 15 sec). Substitution of the bis-Cp rings by five methyl groups augmented the SIA of VDC by 10-fold. The EC₅₀ values (50% inhibitory concentration) for VDC, VMDC, and VPMDC were 7.5 μM, 4.3 μM, and 0.7 μM, respectively. Whereas SIA of vanadocenes was apparent at low micromolar concentrations, the apoptosis-inducing property was evident only at higher micromolar concentrations. The concentrations of VDC, VMDC, and VPMDC required for 50% apoptosis were 49 μM, 67 μM, and 153 μM, and for 50% reduction in sperm ΔΨm were 435 μM, 173 μM, and 124 μM, respectively. Spermicidal activity of vanadocenes was not dependent on the inhibition of ATPase or tyrosine phosphorylation of global and sperm axonemal proteins. Due to the ability of these vanadocene complexes to rapidly generate hydroxyl radicals in the presence of oxidant, our findings provide unprecedented evidence for a novel mechanism of action for spermicidal vanadocenes. The differential concentration-dependent spermicidal and apoptosis-inducing properties of vanadocenes gives them particular utility as a new class of vaginal contraceptives.