The levels and synthesis of histone variants have been directly measured in spermatogonia and in various stages of primary spermatocytes purified from the rat testis. These measurements were made possible by the development of a procedure, employing centrifugal elutriation and density gradient centrifugation, to separate highly enriched populations of such cells from immature rat testes at the early stages of spermatogenesis. The results show a difference in regulation of the synthesis and accumulation of testis-specific histones H1t, TH2A, TH2B, and TH3. TH3 is present and actively synthesized in A and B spermatogonia. The testis-enriched variants, H2A.X and H1a, are also present at their maximal levels in A spermatogonia. No detectable amounts of H1t, and at most, low levels of TH2A and TH2B could be found in spermatogonia. While TH2A and TH2B are already present and actively synthesized in early primary spermatocytes (around the preleptotene stage), H1t does not accumulate until the pachytene stage.