Because most non-melanocytic human skin cancers have p53 mutations, it is unclear whether the aberrant growth of these cancers is simply a result of the abrogation of a p53 downstream mediator, the universal cyclin-dependent kinase inhibitor p21WAF1. To investigate the role of p21WAF1 in human skin carcinogenesis, we studied its regulation in normal and p53-mutated immortalized human keratinocytes. In proliferating human normal keratinocytes (HNK), more wild-type p53 protein (wt p53) was expressed than in growth-arrested differentiating keratinocytes. However, the function of wt p53 as a transcriptional activator of the p21WAF1 gene was suppressed in proliferating keratinocytes. In response to ultraviolet B irradiation, expression of wt p53 increased in proliferating keratinocytes, but p21WAF1 transcriptional activation was not induced. Two isoforms of mdm2 (p57 and p90), which can bind to wt p53 and negatively regulate wt p53 function, were expressed in proliferating HNK, suggesting that mdm2 may play a role in the suppression of wt p53's function in proliferating HNK. Increased expression of p21WAF1 was detected in both Ca(2+)-induced growth-arrested and differentiating HNK, in which the wt p53 expression was down regulated. This reflects the complexity of the p53/p21WAF1 pathways of cell-cycle regulation and differentiation in keratinocytes. No p21WAF1 expression was detected in human immortalized keratinocytes (HaCaT) or in two ras-transformed variants, HaCaT ras I/7 and HaCaT ras II/3, which have two p53 mutations. Retrovirus-mediated expression of p21WAF1 stopped the growth of all these cell types, but expression of wt p53 did not affect the cells' growth properties. p21WAF1 also downregulated human telomerase RNA component mRNA expression in HaCaT cells. This novel function of p21WAF1 partly explains the suppression of telomerase activity by p21WAF1 expression in HaCaT. Taken together, these results are consistent with the idea that p21WAF1 successfully inhibits the growth of non-melanocytic skin cancers, even those with alterations in p53, p21ras, retinoblastoma gene product, and telomerase activity.