Spermatogonial stem cell (SSC) holds great promise in the development of transgenics in farm animals as an alternative to the conventional method involving embryos. One of the big challenges in SSC-mediated transgenesis is the development of simpler method for isolation, purification and culture of SSCs. In the present study, testicular tissue was digested with 1 mg/ml collagenase I and trypsin-EDTA (0.05%, 1mM) at 37°C for 30 min. Following spin, the cells were suspended in complete media and incubated at 37°C and 5% CO2for 3 days. Enrichment of SSC was carried out by adhesion culture onto a gelatine-coated plate. Following culture in DMEM/F12, 10% FBS (foetal bovine serum) and 15 ng/ml of GDNF (glial cell-derived neurotrophic factor) for 7 days, distinct SSC colonies developed which appeared loosely anchored to the surface. The colonies were transferred to a new plate, trypsinized and re-plated on mitomycin C treated Sertoli cell feeder layer for culture for another 7 days. The colonies developed were found positive for alkaline phosphatase activity and appeared as a typical cluster of cells under phase contrast microscope. Thus, a single-step enzymatic digestion involving collagenase I and trypsin-EDTA followed by adhesion culture on gelatine was found to isolate and enrich, respectively, the SSCs from the ovine testis.