Type IV collagen is one of the main constituents of basement membranes, yet it is unknown whether the structural framework at different sites is assembled from one unique type of molecule or whether different type IV collagen molecules exist. To study the composition, chemical identity, and organization of this protein in different organs we have prepared monoclonal antibodies to a type IV collagen preparation from human placenta. Swiss Webster mice were hyperimmunized, and splenic cells were fused with the three different myeloma cell lines SP2/0, NS1, and U1. Type IV collagen-specific hybrids were selected and cloned by limiting dilution and on hard agar. Monoclonal antibodies secreted by two clones were extensively characterized by ELISA-inhibition assay, immunoprecipitation, rotary shadowing, and immunofluorescence techniques. Unlike conventionally raised antibodies in rabbits, both monoclonal antibody reagents show species-specific binding exclusively to native type IV collagen from human placenta but not to a similar preparation from calf lung or to other types of collagen. After heat denaturation of the antigen binding was no longer observed. The M3F7 antibody-binding site is located within the triple helical domain of the type IV molecule, approximately 900 A removed from the amino terminal end as visualized by a metal shadow casting technique. The monoclonal antibody M3F7 precipitates material from pepsin-derived and radiolabeled type IV collagen, and analysis of the polypeptide chains in the immunoprecipitate by sodium dodecyl sulfate polyacrylamide gel electrophoresis suggests that two major fragments are contained in the precipitate, which yield polypeptides of about 100 and 50 kilodaltons. After rotary shadowing of antigen-antibody mixtures native collagen fragments of two different size classes that bind antibody are visualized. One fragment is approximately 1500 A in length, and the other measures about 2700 to 3000 A. The localization of the antigenic site on these fragments suggests that both are generated by pepsin cleavage at a site about 900 A removed from the amino terminal end. In immunofluorescence experiments the monoclonal antibodies stained all basement membranes in kidney, lung, placenta, or skin, suggesting that at least the type IV collagen molecule recognized by these monoclonal antibodies is shared by a variety of vascular and epithelial basement membranes.