Radioactive DNA ([(3)H]cDNA) complementary to the RNA of Mason-Pfizer monkey virus was used in molecular hybridization experiments to demonstrate sequence homology between its viral RNA and RNA of human malignant breast tumors. Hybridization products were analyzed by cesium sulfate equilibrium density centrifugation and hydroxylapatite chromatography. Seven of ten human malignant breast tumors tested contained virus-related RNA when assayed by hydroxylapatite chromatography, whereas no sequence homology was found in RNA from 11 human benign breast tumors or from RNA of normal human spleen, liver, or kidney. Hybridizations between [(3)H]cDNA of the monkey virus and human breast adenocarcinoma RNA showed a C(r)t((1/2)) of about 2 x 10(4). The fidelity of the hybrids formed was indicated by a T(m) of 80.5 degrees .