We examined a candidate tumor suppressor gene on chromosome 9p21, MTS1/CDK4I (multiple tumor suppressor 1/cyclin-dependent kinase 4 inhibitor), which has been found to be mutated frequently in cell lines derived from bladder carcinomas, for somatic mutations in 39 primary bladder cancers by means of SSCP (single-stranded conformational polymorphism) and DNA sequencing. Mutations were detected in two of these carcinomas; one was a 61-base deletion and the other a 1-base deletion. In both cases the homologous allele was missing, indicating that "two-hit" mutation of the MTS1 gene had taken place in these tumors. The results indicated that inactivation of the MTS1 gene is likely to be a contributing factor in some, but not the majority of, bladder cancers.