The physiological basis and immunological significance of thymic enlargement in castrate male animals is not known. We used normal male C57 Bl/6 mice to examine the contribution of in situ thymocyte proliferation to castration-induced enlargement of the thymus. Animals castrated at 8-10 weeks of age were compared to normal intact males. Thymocytes were examined 4-120 days after castration using flow cytometry to determine DNA content and thus the number of cells in active phases of the cell cycle. These properties were examined in unseparated thymocytes and in phenotypic subpopulations defined by expression of CD3, CD4, and CD8. For thymocytes obtained from intact control glands, a mean of 11.0 +/- 1.0% were in active phases of the cell cycle. The percentage of cycling thymocytes was increased to a mean of 22.5 +/- 1.9% in the week after castration (P < 0.001). This change occurred in the absence of significant thymic enlargement. At 8-10 days after castration, thymic weight increased abruptly to a new steady state which was double that of intact controls (78.0 +/- 4.1 vs. 39.1 +/- 2.6 mg; P < 0.001). In these enlarged glands, only 9.9 +/- 0.8% of cells were cycling, which was not significantly different than controls (P > 0.3). Proliferating cells identified in fixed thymus tissue sections after in vivo administration of bromodeoxyuridine were located in the subcapsular cortex and medulla. Analyses of thymocyte subpopulations indicated that most cycling cells had immature phenotypes (CD4+CD8+, CD4-CD8+, and CD3lo or CD3-). Castrate glands studied in the steady state period 8-120 days after surgery contained significantly fewer CD3+ cells than intact controls (P < or = 0.045). The findings suggest an intrathymic role for androgens in affecting generation of the mature T cell repertoire.