The higher prevalence of autoimmune disease among women compared to men suggests that steroids impact immune regulation. To investigate how sex steroids modulate cellular immune function, we conducted a randomized trial in 12 healthy men aged 35-55, treated for 28 days with placebo, a GnRH antagonist, acyline, to induce medical castration, or acyline plus daily testosterone (T) gel to replace serum T, followed by a 28-day recovery period. Serum hormones were measured weekly and peripheral blood lymphocytes (PBLs) were collected biweekly for analyses of thymus-derived lymphocyte (T cell) subtypes and natural killer (NK) cells. Compared to the other groups and to baseline throughout the drug exposure period, men receiving acyline alone had significant reductions in serum T (near or below castrate levels), dihydrotestosterone (DHT), and estradiol (E2) (P<0.05). Medical castration significantly reduced the percentage of CD4⁺CD25⁺ T cells (P<0.05), decreased mitogen-induced CD8⁺ T cell IFN expression, and increased the percentage of NK cells, without affecting the ratio of CD4⁺:CD8⁺ T cells and the expression of NK cell activating receptor NKG2D or homing receptor CXCR1. No changes in immune composition were observed in subjects receiving placebo or acyline with replacement T. These data suggest that T and/or its metabolites may help maintain the physiologic balance of autoimmunity and protective immunity by preserving the number of regulatory T cells and the activation of CD8⁺ T cells. In addition, sex steroids suppress NK cell proliferation. This study supports a complex physiological role for T and/or its metabolites in immune regulation.