Spermatogenesis is a stem-cell dependent process that supplies an indefinite supply of spermatozoa during adult male life. Sperm production is well characterized in rodents and depends on a milieu of endocrine and growth factors, but little is known regarding the regulation of male fertility and spermatogonial stem cells (SSC) in other domestic animals. The objective of the present study was to develop an organ culture system to investigate spermatogenesis in horses. To accomplish this aim we obtained testicular parenchyma from pre- and post-pubertal stallions (n = 3 for each age group) and cultured those tissues upon 0.4-μm membranes in XC medium (DMEM supplemented with 1-, 5- and 10% fetal bovine serum) over a weeklong period with alternate-day media changes. All data sets were evaluated using ANOVA and differences between means were considered significant at P < 0.05. Immunohistochemical analysis of PLZF, GFRA1, and DDX4 expression were used to confirm the identity of undifferentiated spermatogonia, differentiated spermatogonia and meiotic germ cells, whereas GATA4 was used as a marker for Sertoli cell and Leydig cell populations. Histological analysis of fresh and cultured testis tissue indicates the survival of germ-line stem cells and somatic cells populations within morphologically normal seminiferous tubules at 24, 48, and 72 h during the culture period with different levels of serum. Sertoli cell and Leydig cell populations remained stable (P > 0.05) throughout the culture period, but the appearance of pyknotic nuclei in germ and somatic cells observed following 120 h of culture indicates limitations of our approach for long-term tissue maintenance, and this notion is confirmed by TUNEL analysis of apoptosis. Investigating the effect of specific growth factor and hormone treatment combinations on the biological activity and stem cell behavior of undifferentiated spermatogonia is ongoing. Collectively, these results demonstrate successful culture of equine spermatogonial stem cells, and provide a useful ex-vivo model for investigating spermatogenesis in stallions without the costs typically associated with whole-animal experimentation.