Fifty-six primary childhood T-cell acute lymphoblastic leukemia (T-ALL) samples and 17 T-ALL cell lines were examined for mutations and homozygous deletions of the p16/MTS1 gene using polymerase chain reaction single-strand conformation polymorphism and Southern blot analysis. Homozygous deletions were found in 22 primary samples (39%) and in 10 cell lines (59%). In contrast, mutations including small deletions and/or insertions were identified in only 4 primary samples (7%) and in 2 cell lines (12%). Mutations included samples (7%) and in 2 cell lines (12%). Mutations included one nonsense mutation at codon 72, one missense mutation at codon 58, one deletion (29 bp from codon 52-61), one insertion (7 bp into codon 50), and two deletion/insertions (codon 63 and intron 1). Four of the six mutations caused subsequent stop codon and presumably produced truncated p16 protein. Our results suggest that p16 gene alterations are involved in the development of T-ALLs and that the inactivation of the p16 gene occurs mainly through homozygous deletions rather than mutations.