We report cDNA cloning and primary structure of a new metalloproteinase inhibitor (ChIMP-3) produced by chicken embryo fibroblasts. ChIMP-3, formerly called the 21-kDa protein, is one of five ChIMPs (Chicken Inhibitor of MetalloProteinases). In this paper, we report that of the three most abundant ChIMPs, ChIMP-3 and ChIMP-a are extracellular matrix components, whereas ChIMP-2 is found in the media conditioned by the cells. Treatment of ChIMP-3 and ChIMP-a with N-glycosidase-F indicates that ChIMP-a is N-glycosylated whereas ChIMP-3 is not. The deduced amino acid sequence of ChIMP-3 predicts a protein whose properties are consistent with experimental measurements. Analysis of sequence alignments with the two previously described members of the TIMP (tissue inhibitor of metalloproteinases) family, TIMP-1 and TIMP-2, from various species indicates that ChIMP-3 is a related but distinct protein. This conclusion is supported by lack of significant binding with anti-TIMP-1 and anti-TIMP-2 antibodies. Based on these data, its unusual solubility properties, and its exclusive location in the matrix, we propose that ChIMP-3 is a new member of this family of metalloproteinase inhibitors, a TIMP-3.