We employed molecular and immunological techniques to investigate the expression of INSL3, a member of the insulin-like superfamily, in prepubertal testis, postpubertal testes exhibiting normal and disturbed spermatogenesis, and cryptorchid testes of male horses. In addition, the partial cDNA coding sequences of the equine homologue of the human relaxin/INSL3-receptor Lgr8 were determined. Nonradioactive in-situ hybridization with a cRNA probe for equine Insl3 and immunohistochemistry with a specific rabbit INSL3 antiserum localized Insl3 transcripts and immunoreactive INSL3 ligand to Leydig cells in all types of testes investigated. Quantitative polymerase chain reaction analysis revealed a down-regulation of Insl3 and an up-regulation of the relaxin/INSL3-receptor expression in unilateral cryptorchid versus descended testes. Western blot analysis of protein extracts from adult normal and cryptorchid testes and prepubertal testes showed a single immunoreactive band at 14.5 kDa, which correlates with the predicted size of equine proINSL3. Densitometric analysis of Western blot data of adult normal testes revealed significantly stronger expression of immunoreactive proINSL3 as compared to extracts derived from cryptorchid or prepubertal testes. Thus, decreased expression of immunoreactive INSL3 in cryptorchid and prepubertal equine testis is transcriptionally regulated. The detection of transcripts for equine Lgr8 in the testis has identified the testis as a potential target of INSL3.