Though deletion of the long arm of chromosome 6 is one of the most common aberrations in tumors, its targeted gene(s) has not been convincingly identified. Using a functional screening approach, we found that UTRN (which encodes utrophin, a dystrophin-related protein) at 6q24, when expressed in an antisense orientation, induced cellular transformation, consistent with a tumor suppressor role. Northern blot analysis, semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), and gene expression arrays all showed that UTRN expression was downregulated in primary tumors compared with matched normal tissues. Several UTRN neighbor genes were not affected in some tumors with UTRN downregulation, suggesting that UTRN was specifically targeted. RT-PCR, coupled with an in vitro transcription and translation assay, revealed inactivation mutations in 21/62 breast cancers, 4/20 neuroblastomas and 4/15 malignant melanomas. Most of the mutations were deletions involving one or more exons that led to the truncation of utrophin. Splicing errors were found in two cases, and nonsense mutation in one case. Overexpression of a wild-type UTRN in breast cancer cells inhibited tumor cell growth in vitro and reduced their tumor potential in nude mice. Our studies suggest that UTRN is a candidate tumor suppressor gene.