Summary: This study analyzes the morphology and growth characteristics of rabbit ovarian mesothelial (surface epithelial) cells explanted in vitro. Best cell growth and expression of differentiation were obtained using a polystyrene substrate, 100% air, and a nutrient composed of 85% medium 199 and 15% fetal bovine serum. Surface epithelia gave rise to cell outgrowths within 36 h of explantation and formed confluent epithelial monolayers after 1 week. Cells exhibited a marked proliferative activity as indicated by: (a) a labeling index of 80% at day 4, and of 40% at days 10 and 38; (b) a cell doubling time of 30–36 h during the logarithmic phase of growth; and (c) an estimated 24 cell population doublings during 6 weeks. Confluent cultures contained cells cormplexed by desmosomes and tight junctions and displaying numerous microvillintracellular lumina, and focal cilia. Cells organized often into nests and gland like spaces and developed occasional hemicysts. High-density cultures developed also a multilayered configuration, villous processes, epithelial invaginations or crypts, and focal cytological features suggestive of steroidogenesis This study shows that rabbit ovarian surface epithelial cells can be maintained in vitro with a high degree of differentiation. This model is being used to investigate the pathobiology of ovarian surface epithelium.