Using mitomycin C treated 3T3-Swiss fibroblasts as feeder cells, human keratinocytes derived from infant foreskins were subcultured in the presence of trans-retinoic acid (RA) and other retinoids. At 1 microgram/ml (3 x 10(-6) M) and higher RA concentrations plating efficiency was markedly reduced. Addition of retinoids to 1 microgram/ml after colonies were established produced no change in the rate of cell production, but caused differentiated cells to be sloughed earlier than in control cultures. Electron microscopy showed wider extra cellular spaces the contained numerous villi, increased numbers of microvilli at the surfaces of the outermost cells, and decreased number of cell layers all of which were consistent with the observed desquamatory effects of RA. Retinoic acid also extended the time during which cell population increased so that RA treated cultures produced more cells than control cultures over their respective lifetimes. Keratin polypeptides represented a smaller percentage of the total solubilizable protein and more keratin was present as acid soluble prekeratin in postconfluent RA treated cultures. This may be a consequence of early desquamation rather than a decrease in keratin synthesis. No unusual proteins were visible in RA treated cultures by simple sodium dodecylsulfate electrophoresis, nor was there increase in specific activities of three lysosomal enzymes.