Programmed cell death is a controlled process that leads to the elimination of single cells via apoptosis, a mode of cell death with a characteristic morphology. During epidermal differentiation, keratinocytes migrate outward to become terminally differentiated cornified cells in a process involving programmed cell death pathway(s) and apoptosis. The molecular mechanisms regulating epidermal differentiation and apoptosis have not yet been elucidated. Here we show that a mouse keratinocyte cell line, Pam212, undergoes spontaneous apoptosis in culture. Apoptosis of Pam212 cells is demonstrated by both morphology and DNA oligonucleosomal degradation. The expression of bcl-2, a gene implicated in the negative control of apoptosis, was down-regulated in these cells by transfecting a bcl-2-antisense expression vector. The cells that down-regulate bcl-2 expression exhibit enhanced apoptosis and further progress in the epidermal differentiation pathway. We analyzed the expression patterns of several genes that have been implicated in apoptosis in other systems. We show that the mRNA levels of c-myc, c-myb, c-fos, tumor necrosis factors (TNF) alpha and beta, TNF receptors I and II, interleukin 1 alpha, IFN-gamma, and transforming growth factor beta increase in the antisense-transfected cells. We suggest that bcl-2 influences epidermal differentiation in Pam212 keratinocyte cells, and maybe in vivo, by negatively regulating several genes that are involved in apoptosis.