Testosterone administration to female mice for 25 days produced a 70% increase in total kidney protein in both A/J and C57BL/6J mice. This is in contrast to the known androgen-responsive proteins, such as beta-glucuronidase and alcohol dehydrogenase, which each represent less than 1% of the total kidney proteins even after maximum stimulation. To investigate this discrepancy, we initiated a study to identify major proteins which increase with androgen treatment. Three new cytoplasmic proteins designated T1, T2, and T3 were found in different subcellular fractions of both A/J and C57BL/6J mice. T1 (43,000 daltons) and T2 (60,000 daltons) were found in the mitochondrial-lysosomal fraction of 25-day androgen-treated mice. T3 (54,000 daltons) was found in the microsomal fraction. Each of these proteins increased several-fold during androgen treatment, so that they were easily identified using sodium dodecyl sulfate polyacrylamide gel electrophoresis. By contrast, no major changes were noted in the soluble proteins. A nonhistone chromosomal protein of 54,000 mol wt (T4) was found in chromatin preparations from androgen-stimulated A/J mice. Additional studies with androgen-insensitive Tfm/Y mice and with various hormones indicated that stimulation of the T proteins was dependent on androgenic steroid and a functional androgen receptor.