A cDNA encoding a novel protein has been previously isolated from two independent sources: melanoma cell cultures and chondrocytes. The protein from human melanoma cell lines and tumors is called melanoma inhibitory activity (MIA) (Blesch et al.  Cancer Res. 54:5695-5701) and the protein from primary bovine chondrocytes and cartilaginous tissues is called cartilage-derived retinoic acid-sensitive protein (CD-RAP) (Dietz and Sandell  J. Biol. Chem. 271:3311-3316). In order to investigate the gene regulation and function of CD-RAP/MIA, the mouse gene locus was isolated and analyzed. Developmental expression was determined by in situ hybridization to mouse embryos. Expression was limited to cartilaginous tissues and was initiated with the advent of chondrogenesis, remaining abundant throughout development. The mouse gene was isolated and sequenced from a 129Sv library and sequenced directly from an additional strain, B6C3Fe. The mouse CD-RAP/MIA gene is 1.5 kbp and consists of four exons. The promoter sequence of the gene contains many potential regulatory domains including 8 basic helix-loop-helix protein-binding domains and an AT-rich domain, both motifs shown to be present in the cartilage-specific enhancer of the type II procollagen gene. Other potential cis-acting motifs include binding sites for GATA-1, NF-IL6, PEA3, w-elements, NF kappa B, Zeste and Sp1. The gene, called cdrap, was localized to the end of an arm of chromosome 7 at the same site as the transforming growth factor beta 1 (Tgf-beta 1) and the glucose phosphate isomerase 1 (Gpi 1) genes. Potential mouse mutants that mapped to the same region of chromosome 7 were identified. Two of the potential mutants with skeletal phenotypes were sequenced, pudgy (pu) and extra toes with spotting (XsJ); however, no mutations were found in the coding sequence. To determine whether CD-RAP/MIA is associated with tumors of cartilage, mRNAs from a variety of rodent tissues and cell lines were screened. Expression was detected in a rodent tumor, the Swarm rat chondrosarcoma and a chondrosarcoma cell line derived from it, but not in other tissues or tumors of non-cartilage origin. Immunolocalization revealed CD-RAP/MIA protein localized in cartilage only. These results show that the normal expression of CD-RAP/MIA is limited to cartilage; however, pathologically, it is expressed both in melanoma and chondrosarcoma. The restricted expression of CD-RAP/MIA may provide an opportunity to monitor cartilage metabolic activity as well as the tumor activity of melanoma and chondrosarcoma.