BACKGROUND: Spermatogonial expansion in man and non-human primates has been studied for decades. Controversy persists about the cell type representing the testicular stem cell and the exact kinetics of spermatogonial proliferation. We recently determined the starting point of spermatogenesis and proposed a model for clonal expansion of spermatogonia in adult macaques. Here we want to confirm the initiation event, study and compare the details of the kinetics of spermatogonial expansion in vivo and in vitro, and characterize a population of A spermatogonia acting as testicular stem cells. METHODS and RESULTS: We localized BrdU-positive spermatogonia in whole mounts and sections of adult rhesus monkey testes. Culture of testicular tissue was used to determine the expansion and differentiation of premeiotic germ cells. We confirm that A_pale spermatogonia divide equally at stage VII and produce two types of progeny after mitosis at stage IX of the seminiferous cycle following defined clonal patterns. Small numbers of proliferating single A spermatogonia exist which present a population of label-retaining cells. CONCLUSIONS: In the rhesus monkey the population of A_pale spermatogonia cycle continuously and initiate spermatogenesis by a self-renewing division at stage VII of the seminiferous epithelial cycle. Rarely dividing single A spermatogonia exist which potentially are the male germline stem cells in the primate testis.