Cytochrome P450 CYP1B1 is a recently identified member of the CYP1 P450 family. We have shown that this P450 displays increased expression in several types of human cancer, indicating that CYP1B1 is a potential tumor biomarker. In this study we developed monoclonal antibodies (MAbs) to CYP1B1 that are effective on formalin-fixed, paraffin-embedded tissue sections and investigated the presence of CYP1B1 in a series of primary breast cancers. The MAbs were generated using a synthetic peptide coupled to carrier protein as the immunogen. The MAbs specifically recognized CYP1B1 and did not recognize either CYP1A1 or CYP1A2, related CYP1 forms. The MAbs were tested by immunohistochemistry and were found to be effective on formalin-fixed, paraffin-embedded tissue sections. The majority of breast cancers showed positive immunoreactivity for CYP1B1, and in each case CYP1B1 was specifically localized to tumor cells. The presence of CYP1B1 in breast cancer cells is likely to contribute to their metabolism of estradiol because CYP1B1 is a specific estradiol hydroxylase. (J Histochem Cytochem 47:1457-1464, 1999)