A B S T R A C T The metabolism of "C-labeled testosterone by cultured human fibroblasts and amniotic fluid cells was investigated. Radiolabeled testosterone was incubated with the cultured cells for 48 hr, and the labeled metabolites present in the medium were subsequently identified. The major metabolic products of testosterone formed by cultured fibroblasts were A'-androstenedione, dihydrotestosterone, androsterone, and androstanediol. The amount of testosterone metabolized through each of two pathways was calculated and used to form a ratio designated the 17f-hydroxyl/17-ketonic ratio. Fibroblasts from normal male and female children and adult females had high 17P-hydroxyl/17-ketonic ratios indicating testosterone metabolism occurred primarily through the 17P-hydroxyl pathway. There was a change in the pattern of testosterone metabolism with age in males, i.e., adult males had much lower 17#-hydroxyl/17-ketonic ratios than did male children.The testosterone metabolism of fibroblast cultures derived from three children with testicular feminization and their mothers was compared to normal age and sexmatched controls. Fibroblasts of children with testicular feminization metabolized testosterone predominantly through the 17-ketonic pathway and manifested a pattern of testosterone metabolism distinctly different from their sex and age matched controls. The mothers of children with testicular feminization could be distinguished from normal females by their much lower 17#-hydroxyl/17-ketonic ratios. The much lower amounts of dihydrotestosterone and androstanediol produced by fibroblasts from patients with testicular feminization as