The effect of norethisterone, norethisterone acetate, ethinylestradiol and of a cyanoketone on the activity of HSD have been studied. Rat Leydig cells and extracts of Pseudomonas testosteroni were used as enzyme sources. Conversion of [3H]DHEA to A and [3H]pregnenolone to progesterone were used for the assay of enzyme activity. Km values for each substrate for Leydig cells and for bacterial enzyme were: 1.5 x 10(-5) mol l-1 and 6.0 x 10(-5) mol l-1 DHEA; 1.3 x 10(-5) mol and 7.7 x 10(-5) mol l-1 pregnenolone. For investigation of inhibition [3H]DHEA was used as substrate and apparent Ki values were calculated by Dixon plot analysis. In both systems norethisterone showed no inhibition of the enzyme. Norethisterone acetate proved also to be not inhibitory; only residual affinity to the enzyme was noted (Ki = 8.0 x 10(-5) mol l-1: Leydig cell preparation; Ki = 9.4 x 10(-5) mol l-1: bacterial enzyme). Different result sin the two systems were obtained for ethinylestradiol and for a combination with norethisterone acetate. Ethinylestradiol or the combination with the progestin (Ki = 0.5 x 10(-5) mol; 1.4 x 10(-5) mol l-1) showed noteworthy inhibition of the bacterial enzyme but only weak competition for binding to the active site of the enzyme in rat Leydig cells (Ki = 6.0 x 10(-5) mol l-1; 8.3 x 10(-5) mol l-1). It is concluded that there are greater differences between the two enzymes than considered previously. As expected, cyanoketone proved to be the most effective inhibitor in both systems. The most important result seems to be, that norethisterone does not inhibit HSD. This suggests that there is no causal relationship between hypospadias and progestins administered to females during pregnancy.