The p16(INK4a) and p14(ARF) tumor suppressor genes (TSGs) are encoded within the CDKN2A locus on chromosome 9p21 and function as cell cycle regulatory proteins in the p53 and RB pathways. Inactivation of these genes by genetic and epigenetic changes has been described in some human cancers, but their importance in cutaneous squamous cell carcinoma (SCC) has not been established. Our detailed examination of 40 cutaneous SCC revealed loss of heterozygosity of 9p21 markers in 32.5% of cases. Mutational analysis confirmed five point mutations in four of 40 SCCs. These mutations changed the amino acid sequence of p16(INK4a) in four tumors and p14(ARF) in three tumors. Promoter methylation of p16(INK4a) and p14(ARF) was detected in 13 of 36 (36%) and 16 of 38 (42%) cases, respectively. Absent protein expression was confirmed by immunohistochemistry in 13 of 16 (82%) of the tumors with biallelic inactivating events. Overall, the frequency of 9p21 alterations was 76% and for both p16(INK4a) and p14(ARF), promoter methylation is the commonest mechanism of gene inactivation. Alterations at this locus were significantly more common in tumors from immunocompetent compared with immunosuppressed individuals. These data confirm the importance of inactivation of p16(INK4a) and p14(ARF) TSGs in the pathogenesis of cutaneous SCCs.