Human epidermal keratinocytes may be isolated in high yield from 0.1 mm keratotome sections of adult skin by short-term trypsin release. When plated on a collagen-coated plastic surface or on a collagen gel, keratinocytes attach with high efficiencies (greater than 70%) and form confluent, stratified cultures. Cell populations of predominantly basal cells produce proliferative primary cell cultures while populations of basal cells and malpighian cells result in nonproliferative primary cultures. Both nonproliferative and proliferative primary cultures may be subcultured on collagen gels following dispersion by trypsin and EDTA. Methotrexate strongly inhibits proliferative keratinocytes at low concentrations (0.1 microgram/ml) but has no cytotoxic effect on non-proliferative cells. L-serine and dexamethasone increase the multiplication rate of both primary and subcultured human keratinocytes.