The steroidogenic enzymes present in boar testicular tissue use steroid sulfates as substrates. Pregnenolone sulfate, doubly labeled with 3H in the nucleus and 35S, was incubated with the microsomal fraction of boar testicular tissue and a reduced TPN generating system and 17-hydroxypregnenolone sulfate and dehydroisoandrosterone sulfate were isolated. These products had the same 3H to 35S ratio as the substrate demonstrating that the conversions had taken place with the sulfate group intact. Testicular 17-hydroxylase and C-17,20-desmolase can convert .DELTA.5-3.beta.-yl sulfates into products which still contain the sulfate group at C-3. An unusual steroid sulfate, doubly labeled 5,16-androstadien-3.beta.-yl sulfate, was also isolated demonstrating that such an olefin can be biosynthesized via steroid sulfate pathways. In a 2nd experiment small amounts of [3H]-21-hydroxypregnenolone sulfate were isolated from the incubation of [3H]pregnenolone sulfate with microsomes from boar testes. The isolation of the 21-hydroxylated steroid sulfate is taken as support for the hypothesis that .DELTA.16 steroids are biosynthesized from their C21 precursors by events that are initiated by oxygenation at C-21.