We have investigated phase II activation of the food-derived mutagen 2-hydroxyamino-1-methyl-6-phenyl[4,5-b]pyridine (N-OH-PhIP) by cytosolic acetyltransferase, sulfotransferase, and tRNA synthetase/kinase enzymes from human breast tissue. Cytosol from homogenates of mammary gland tissue obtained from breast-reduction surgery or mastectomy was incubated with and without enzyme-specific cofactors, and mutagen binding of calf thymus DNA was quantified by 32P-postlabeling. In addition, microsomal fractions of mammary epithelial cells from some individuals were examined for prostaglandin H synthetase activation of N-OH-PhIP. Our results show that all four enzymes can participate in activating N-OH-PhIP, thus inducing PhIP-DNA adduct formation in human mammary cells. However, not all individuals exhibited all these activities; instead each individual showed a combination of one or more activation pathways. The present findings demonstrate that the human mammary gland has the capacity to metabolically activate a dietary mutagen by several enzyme systems, including acetyltransferase, sulfotransferase, tRNA synthetase/kinase, and prostaglandin hydroperoxidase catalysis.