Targeted gene deletion of insulin-like growth factor-I (IGF-I) results in diminished numbers of Leydig cells (LCs) and lower circulating testosterone (T) levels in adult males. The impact of endogenous IGF-I withdrawal on proliferation (labeling index, LI) and differentiation of LCs was investigated, testing for restorative effects of IGF-I replacement and/or LH stimulation. With IGF-I replacement in mutant mice, LIs increased more than 200% (P < 0.05). LC numbers were also increased by 200%, whereas the numbers of intermediate cell progenitors (PLCs) were unchanged compared to mutant vehicle controls. LIs of PLCs in wild-type males increased by 200% after LH stimulation, and LC numbers increased by 50% compared to vehicle-treated controls (P < 0.05). In contrast, there was no effect of LH on LI in mutant mice, but LC numbers still increased by 30% (P < 0.05). Additive effects on LI and cell numbers were observed in response to IGF-I plus LH in mutants, implying that the two hormones use separate signaling pathways. Serum T and LH levels in wild-type and mutant males were equivalent. Exogenous LH increased T production 8-fold in wild-type males (P < 0.01). In mutant mice, neither LH stimulation nor IGF-I alone affected serum T levels, but IGF-I plus LH stimulation increased serum T 2-fold (P < 0.05). These data support the conclusions that 1) IGF-I is a critical autocrine and/or paracrine factor in the control of adult LC numbers and function; and 2) LH is not a direct mitogenic factor for LCs, and acts in part through IGF-I to stimulate proliferative activity.