In humans. Leydig cells are present during the first 6 months of postnatal life and they regress thereafter. We have described that basal (B) secretion of testosterone (T) in human testicular cells in culture is higher between 1 and 7 months (M) of age than between 8 and 36 M. Furthermore. T was stimulated by LH, FSH and GH only in the younger group (Ped Res 1995, in press). In the present study of primary cultures of human testicular cells obtained at necropsy, we have evaluated the activity of 3 enzymes of testicular steroidogenesis, 17,20 -DES, 3βHSD and 17β HSD, by correlating the production of T (the final product of steroidogenesis) with that of 17OH-progesterone (17OHP) (for 17,20-DES and 17βHSD), dehydroepiandrosterone (DHEA) (for 3βHSD and 17βHSD) and androstenedione (A) (for 17βHSD). Activities were estimated from the slopes (SL) of regression lines. Levels of T, 17OHP, DHEA and A were determined by RIA in conditioned media of day 6 of culture. The following groups (G) were defined, G1B, aged 1 to 7 months (n=8), G1 ST same age but under chronic stimulation (ST) with LH (10 ng/ml), FSH (2 ng/ml) or GH (50 ng/ml) and G2B, aged 8 to 36 M (n=7). Between 17OHP and T, SL was 1.95(r=0.73, p=0.0001) in G1B and 6.99 (r=0.97, p=0.0001) in G2B These two SL were significatively different (p<0.0005) but no difference was observed between G1B and G1ST. Between DHEA and T, SL was 0.60 (r=0.967 p<0.0001) in G2B but no significant correlation was observed in G1B nor in G1ST. Between A and T, SL were similar, 0.48 (r=0.78, p=0.0001) and 1.09 (r=0.56, p=0.02) in G1B and G1ST, respectively, but different (p<0.005) from G2B, 2.18 (r=0.99, p=0.0001). Furthermore, in G1B and in G2B SL between A and T were significantly lower than between 17OHP and T (p<0.005 in both). It is concluded that in immature human testicular cells, the activities of 17,20 DES, 3βHSD and 17βHSD do not change after trophic stimulation. The absence of significant correlation between DHEA and T suggests that 3βHSD is very active. Since SL are higher in G2B in the pre-Leydig cells of the regressed testis. the 3 enzymes analysed are probably low. Finally, the fact that SL between A and T is smaller than between 17OHP and T suggests that 17,20-DES is a limiting enzyme activity in the steroidogenesis of the immature human testis.