The activities of enzymes which synthesize and metabolize catecholestrogens were studied in biopsy samples of human breast neoplasms. Estrogen 2-hydroxylase, a cytochrome P-450-dependent enzyme, was present in benign and malignant neoplasms but not in normal breast tissue. Catechol O-methyltransferase activity was present in all samples examined and was significantly higher in malignant tumors [549 .+-. 31 (S.E.) pmol/20 min per mg protein] than in benign neoplasms (226 .+-. 41 pmol/20 min per mg protein) or in normal breast tissue (133 .+-. 28 pmol/20 min per mg protein). There was no correlation between estrogen 2-hydroxylase and catechol O-methyltransferase activities. The enzymes responsible for the synthesis and metabolism of catecholestrogens are present in some breast tumor specimens, suggesting that in such tissues these metabolites may be formed in vivo.