Netrins, a family of laminin-related secreted proteins, have critical roles in axon guidance and cell migration during development. The deleted in colorectal cancer (DCC) protein has been implicated as a netrin-1 receptor component. The expression and function of netrins in adult tissues remain unknown, and direct interaction of netrin-1 with DCC has not been demonstrated. We cloned the human netrin-1 (NTN1L) gene, mapped it to chromosome 17p12-13, and found that it encodes a 604 amino acid protein with 98% identity to mouse netrin-1 and 50% identity with the Caenorhabditis elegans UNC-6 protein. NTN1L transcripts were detected in essentially all normal adult tissues studied, and markedly reduced or absent NTN1L expression was seen in approximately 50% of brain tumors and neuroblastomas. In one neuroblastoma, missense mutations at highly conserved NTN1L codons were found. Netrin-1 protein could be cross-linked to DCC protein on the cell surface, but it did not immunoprecipitate with DCC in the absence of cross-linking and it failed to bind to a soluble fusion protein containing the entire DCC extracellular domain. Our findings demonstrating NTN1L loss of expression and mutations suggest that NTN1L alterations may contribute to the development of some cancers. Furthermore, the binding of netrin-1 to DCC appears to depend on the presence of a coreceptor or accessory proteins.