The possible interactions between naturally occurring steroids and three enzymes of the biosynthetic pathways of 16-androstenes and androgens in the boar testis were investigated. High concentrations (1 mmol/1) of several steroids reduced the production of 5,16-androstadien-3 beta-ol when a microsomal fraction of boar testis was incubated with pregnenolone at pH (the pH optimum of this reaction was found to be ). When some of these inhibitors were investigated in more detail using Lineweaver-Burk analyses, the apparent inhibition constants, Kv increased in value with increasing concentrations of inhibitors. When testosterone was added to 5,16-androstadien-3 beta-ol synthetase assays, the apparent Ki for mumol testosterone/1 was mumol/1 whereas those for , and mumol testosterone/1 were , and mumol/1 respectively. The apparent Michaelis constant, Km, of the reaction was mumol/1. Similar results were obtained when oestrone, 17 alpha-hydroxyprogesterone and 4,16-androstadien-3-one were added as effectors. At physiological concentrations, these steroids would not affect the biosynthesis of 5,16-androstadien-3 beta-ol in vivo. Similarly, both 5 alpha-androst-16-en-3 beta-ol, quantitatively the most important 16-androstene in the boar testis and 5,16-androstadien-3 beta-ol were examined for their effects on the 17 alpha-hydroxy-C21-steroid, C-17,20 lyase (C-17,20 lyase) and 17 beta-hydroxysteroid dehydrogenase (17 beta-OHSDH) enzymes of androgen biosynthesis. Neither of these enzymes was affected by the 16-androstene steroids even at concentrations of 100 mumol/1, and the apparent Km values were and mumol/1 for C-17,20 lyase and 17 beta-OHSDH respectively. This lack of interaction between these pathways implies that the high levels of 16-androstene steroids produced by the testis will not interfere with androgen production, and also that the two side-chain cleavage steps from C21 precursors to C19 steroids are catalysed by independent systems.