The INK4A locus on human chromosome 9p21 encodes two genes that have been implicated in replicative senescence and tumor suppression, p16INK4A and p14ARF. In contrast to p16INK4A, which is up-regulated to high levels, we were unable to detect p14ARF protein in senescent human keratinocytes. Also, p53, an established target of p14ARF, did not increase, suggesting that p14ARF is not instrumental in human keratinocyte senescence. In neoplastic keratinocyte cultures, p16INK4A inactivation was invariably associated with the immortal phenotype, and there was evidence for the inactivation of p16INK4A, independent of p14ARF, in 6 of 10 lines that lacked large homozygous deletions. In contrast, we failed to detect exon 1beta mutations or p16INK4A-independent deletions. These results emphasize the previously proposed role for p16INK4A in human keratinocyte senescence but do not rule out a supporting role for p14ARF inactivation.