We have produced a panel of monoclonal antibodies directed against a dimethylbenzanthracene-induced murine mammary tumor. Five rat-mouse hybridomas produced antibodies that bound to some murine mammary tumors, but not to normal renal adherent cells, lymphocytes, 3T3 fibroblasts, red blood cells, or mammary gland. One of these antibodies, designated AMT8, was selected for further evaluation based on its relatively strong reactivity, as determined by immunofluorescence. Indirect immunofluorescent studies on frozen histological tissue sections and quantitative immunofluorescent binding studies on cultured normal and tumor cells revealed that AMT8 was bound to certain murine mammary tumors and their preneoplastic hyperplastic nodules, but not to normal murine organs including normal mammary glands. Two tumors and their hyperplastic alveolar nodule counterparts that contained the antigen recognized by AMT8 did not express functional estrogen and progesterone receptors, indicating that antigen expression was not dependent on functional receptors. The antigen recognized did not cap, was found to modulate slowly, and was reexpressed in the presence of excess AMT8. From these findings, we conclude that AMT8 may prove to be a valuable tool for the study of early mammary tumorigenesis.