Current knowledge regarding the molecular composition of extracellular matrices in the glomerulus does not explain how these components interact to form stable three-dimensional structures. The recent recognition that short-chain collagens such as type VIII collagen function as molecular bridges in some nonrenal tissues has raised the possibility that such molecules may serve a similar function in the glomerulus. We have recently shown that cultured rat mesangial cells synthesize and secrete several short-chain collagenous proteins, one of which has properties similar to alpha 1-VIII collagen. In the present study we have isolated a rat mesangial cell alpha 1-VIII collagen cDNA clone, the sequence of which is 81% homologous to mouse alpha 1-VIII collagen. We used this cDNA to determine that alpha 1-VIII collagen mRNA is expressed in rat renal cortex and in cultured glomerular mesangial, epithelial, and endothelial cells. Additionally, we demonstrated that alpha 1-VIII collagen is secreted by cultured mesangial cells as an 80-kDa translation product. By immunocytochemistry, alpha 1-VIII collagen localized to the media of large intrarenal arteries and to the capillary loops and the mesangium of normal rat kidney. These results indicate that type VIII collagen is a normal constituent of the rat glomerulus as well as large intrarenal arteries. We speculate that type VIII collagen may function in part to determine the three-dimensional organization of the subendothelial and mesangial matrices.