We determined the concentration of steroids in blood plasma of CBF1 male mice and the steroidogenic potential of the mouse testis. Steroid secretion rates are based on measuring selected C18, C19, and C21 steroids in the venous effluent of testes perfused with a defined medium containing luteinizing hormone. Steroids were isolated by thin-layer and/or column chromatography and quantified by radioimmunoassay. These include pregnenolone, 17 alpha-hydroxypregnenolone, dehydroepiandrosterone, androstenediol, progesterone, 17 alpha-hydroxyprogesterone, androstenedione, testosterone, dihydrotestosterone, 3 alpha-androstanediol, and 3 beta-androstanediol. Testosterone is the primary steroid secreted by mouse testes perfused in vitro and is the chief androgen present in blood plasma. Pregnenolone, an obligatory intermediate in steroid synthesis, is converted to testosterone via two separate steroidogenic pathways in approximately equal proportions. This is unlike other species in which testosterone biosynthesis proceeds preferentially via either the delta 4 or the delta 5 pathway. Our results, taken together, provide the first comprehensive assessment of Leydig cell steroidogenic activity in the mouse, demonstrate putative enzymatic pathways subserving androgen biosynthesis, and establish the predominant steroids in the peripheral circulation of adult mice.