The results of a thymidine incorporation assay were compared with direct measurement of cell number in assessment of proliferative growth of human keratinocytes in monolayer culture. Keratinocytes were cultured in supplemented MCDB 153 medium in 0.1 mM Ca2+, and plated in 24-well trays. The ability of insulin, placental extract, and epidermal growth factor to enhance growth and thymidine incorporation were compared. Autoradiography was performed to determine the percentage of cells with labeled nuclei. Epidermal growth factor increased thymidine incorporation under the conditions of the assay, and placental extract increased incorporation by up to 50-fold, since the control cells plated in the absence of epidermal growth factor and other growth factors survived but proliferated minimally. Both cell number and thymidine incorporation showed similar concentration dependence upon insulin and placental extract. If placental extract was added to cells plated 28 h earlier, incorporation was maximal after 17 h in the presence of the extract. If cells were plated in the presence of the extract, 85% of nuclei were shown by autoradiography to be labeled after 23 h, but only 24% of nuclei were labeled in the absence of the extract. A plating density of 10(4) cells/2-cm2 well was optimal. The assay permits rapid identification of growth-promoting fractions without prolonged growth periods, and is a valid indicator of these agents in keratinocyte cultures.