3βHSD is an essential enzyme in the biosynthesis of steroid hormones. Little information is available on the regulation of the 3βHSD gene in the human testis. We have studied the role of IGF-I and of insulin in the expression of 3βHSD in mRNA in primary culture of mixed testicular cells collected at necropsy from a 15.5-year-old boy with sexual development and no endocrinological disorder. On day 6 of culture, the levels of testosterone(T). Δ4 androstenedione (A) dehydroepiandrosterone (DHEA) and 17OH progesterone (17OHP4) in conditioned medium were determined by RIA in basal conditions and under chronic stimulation with IGF-I (500 ng/ml) or insulin (50 ng/ml). On the same day, 3βHSD mRNA was analyzed by Northern Blot in the cells. A complete fragment of hp3βHSD 63 cDNA. labelled with P32, was used as a probe. The same membrane was washed and rehybridized with a 244 bpβ-active cDNA probe as an internal control. Mean ±SD (n=3 basal levels of T, A, DHEA and 17OHP4 were respectively: 34.3±0.66, 75.2±10.4, 119±29.5 and 119±14.8 pmol/106 cells.24 hs. Only under IGF-I stimulation the 3 steroids were significantly increased: T 159±2.5, A 216±10.5 and 17OHP4 237±6.2 (p< 0.05, ANOVA). The 1.7 Kb characteristic band of 3βHSD mRNA was only detected in the presence of IGF-I. It is concluded that during pubertal development of the human testis IGF-I stimulates steroidogenesis regulating the expression of the 3βHSD gene. The fact that insulin did not stimulate steroidogenesis neither it induced expression of 3βHSD mRNA suggests that the effect of IGF-I was mediated through the IGF-I receptor and not through the insulin receptor.