Although epithelial-like somatic cells have been previously isolated from semen, cell proliferation rates were low. Culture of whole semen samples resulted in loss of potentially valuable spermatozoa. The aims of the present study were to: (1) isolate somatic cells from semen, while preserving sperm viability, and (2) optimize in vitro culture conditions for semen-derived epithelial cells. Density gradient centrifugation of washed ejaculates of two rams (Ovis aries) (n = 24) and one eland bull (Taurotragus oryx) (n = 4) was performed using a three-layer discontinuous Percoll column consisting of 90% (P-90), 50% (P-50), and 20% (P-20) Percoll. In vitro culture and Trypan Blue staining indicated that live somatic cells settled in the P-20 layer. Nonmotile spermatozoa were recovered at the P-50 and P-90 interfaces, whereas motile spermatozoa were collected in the pellet from the P-90 layer. Subsequently, somatic cells isolated from the P-20 layer were plated either on inactivated 3T3 mouse embryonic fibroblast feeder layers, collagen-coated plates with 3T3 feeder cell inserts, or on collagen-coated plates. Initial somatic cell plating was similar among treatments, but proliferation significantly increased when cocultured with 3T3 cells (feeder or insert). Furthermore, two different types of epithelial cells were obtained. The exact origin of the cells in the male reproduction system is uncertain and probably variable. The present method of cell isolation and in vitro culture may be of value for preserving endangered species. Specifically, cells isolated and cultured from cryopreserved semen of nonliving males could be used for producing embryos by somatic cell nuclear transfer.