A method to isolate and to serially cultivate rabbit skin epithelial cells from adult trunk skin has been developed. Using a collagen gel as substrate and trypsin and EDTA to dissociate cells, nonproliferative primary cultures of rabbit cells may be converted to proliferative populations, and at least 3 serial passages achieved. In the presence of large concentrations of methotrexate (up to 1000 microgram/ml), epithelial cells in primary culture show no decrease in their ability to attach, spread, or keratinize. Following conversion to proliferative populations by trypsin and EDTA low concentrations of methotrexate (1 microgram/ml) are strongly cytotoxic. When the incorporation of 32PO4(3-) and 3H-thymidine into DNA of proliferative and nonproliferative cells is compared, the incorporation of 32PO4(3-), but not that of 3H-thymidine, correlates with changes in cell number and DNA content. In both primary and serially cultivated cells, L-serine is required for optimal growth.