We have previously suggested that a gene mapping to chromosome 9p21 could contribute to replicative senescence and suppress cullular immortality in squamous neoplasia. Two candidate genes, the cyclin D1/cyclindependent kinase inhibitors CDKN2A/p16INK4A (p16) and CDKN2B/p15INK4B (p15) have now been identified in this region and we show here that p16 is upregulated when normal human keratinocytes undergo replicative senescence but not when they undergo differentiation. Furthermore, all of 19 immortal neoplastic keratinocyte head and neck lines, including nine showing loss of heterozygosity (LOH) at 9p21, showed undetectable p16 expression, whereas five of six senscent neoplastic cultures showed normal levels of expression. The retinoblastoma protein (pRb) appeared functional in all the cell lines and cultures examined. The mechanism of p16 inactivation appeared to be transcriptional silencing in 10 of 18 lines and homozygous deletions in the rest. Treatment of two of the immortal cell lines which had transcriptionally silent wild type p16 genes with 5aza-2deoxycytidine resulted in the re-expression of p16, thus implicating DNA methylation as one mechanism of transcriptional silencing in the immortal SCC-HN lines. We observed no cases of p16 point mutation. In contrast, the p15 gene was rarely transcriptionally silent and was not deleted in any of the cell lines which showed p16 deletions. Our results show that p16 dysfunction correlates strongly with keratinocyte immortalisation but less strongly with the stage of tumour progression. P16 dysfunction was not related to the neoplastic state or the length of time spent in vitro. The results also suggest that p16 but not p15 is involved in the keratinocyte replicative senescence programme. However, two neoplastic cell cultures which lacked p16 expression were still mortal, suggesting that the loss of p16 is a necessary but insufficient condition for human keratinocyte immortality.