We have characterized the cell and tissue binding specificity of a newly generated monoclonal antibody, Mab Ku-1, which shows selective reactivity with rat macrophages and Kupffer cells. The hybridoma secreting Mab Ku-1 was constructed by fusion of 8653 myeloma cells with spleen cells isolated from a mouse immunized with nonparenchymal liver cells coated with antihepatocyte antibodies. When binding was assessed by indirect immunofluorescence on frozen sections from normal liver tissue, Mab Ku-1 showed strong reactivity with Kupffer cells but was unreactive with hepatocytes, endothelial cells, bile ducts or lymphocytes. Both resident and activated macrophages bound Mab Ku-1. Reactivity in other tissues was compatible with specificity for macrophages. In the gut, scattered cells in the lamina propria were positive, whereas epithelial cells were negative. Individual cells in the lung were reactive. In the spleen, cells in the red pulp peripheral to germinal centers bound antibody. Reactivity of Mab Ku-1 to isolated Kupffer cells correlated with endogenous peroxidase activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of components immunoprecipitated by Mab Ku-1 from detergent lysates of Kupffer cells biosynthetically labeled with 35S-cysteine and 35S-methionine demonstrated that the reactive antigen was a peptide with an apparent molecular weight of 107 kD. This rat macrophage-reactive monoclonal antibody is a useful marker for identification of macrophage populations in tissue as well as in isolated cell populations.