Cryptorchidism is a common reproductive abnormality, possibly resulting from abnormal hormone production/action by the fetal testis. Insulin-like factor 3 (Insl3) is thought to be involved in gubernaculum development and transabdominal testicular descent, but its importance is unclear, due partly to lack of suitable Insl3 antibodies. We generated (by genetic immunization) and validated a novel antirat Insl3 antibody, which we used to characterize immunoexpression of Insl3 in rat Leydig cells (LCs) from fetal life until adulthood and its relationship to cryptorchidism. Immunoexpression was strong on embryonic day (E) 17.5 and E19.5 and from 35 d of age onward but weak from E21.5 until puberty. Because in utero exposure to di (n-butyl) phthalate (DBP) induces cryptorchidism and suppresses Insl3 gene expression, we investigated Insl3 protein expression in fetal and adult rats exposed to 500 mg/kg.d DBP from E13.5 to E21.5. Expression on E17.5 and E19.5 decreased dramatically after DBP exposure, but there was no consistent correlation between this suppression and abnormal testis position. We also compared expression of Insl3 and P450 side-chain cleavage enzyme in fetal testes from rats exposed in utero to DBP or flutamide (50 mg/kg.d). DBP treatment suppressed expression of both P450 side-chain cleavage enzyme and Insl3 at E19.5, but flutamide exposure had no effect on either protein, demonstrating that Insl3 expression in fetal rat LCs is not androgen regulated. In adult rats, Insl3 expression was suppressed in 80% of cryptorchid and 50% of scrotal testes from rats exposed to DBP, suggesting that prenatal DBP exposure also leads to maldevelopment/malfunction of the adult LC population in some animals. ; Chris McKinnell, Richard M. Sharpe, Kim Mahood, Nina Hallmark, Hayley Scott, Richard Ivell, Christophe Staub, Bernard Jégou, Friedrich Haag, Friedrich Koch-Nolte, and Stefan Hartung