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The period of Leydig cell hyperplasia (14–18 weeks gestation) in human fetal testis is crucial for normal gonad development. We have studied the spatio-temporal distribution of key developmental and functional markers in human fetal testis between 13–19 weeks gestation. Proliferating cell nuclear antigen-positive cells were immunolocalized to both interstitium and tubules. Image analysis confirmed an increase in positive interstitial cells during Leydig cell hyperplasia (P < 0.05). c-Myc was localized to the interstitium with no gestational changes. The steroidogenic enzymes 3β-hydroxysteroid dehydrogenase (protein) and cytochrome P450 17α-hydroxylase/C17–20-lyase (P450c17; messenger ribonucleic acid and protein) were confined to the Leydig cells. The number of immunopositive cells increased between 13 and 19 weeks (P < 0.001). P450c17 mRNA (in situ hybridization) and protein were localized to the same population of interstitial Leydig cells. Androgen receptor and Bcl-2 protein (anti-apoptotic) were gradu...