Alkyltransferase (AGT) repairs alkylation at O6-guanine in DNA and is a major determinant of susceptibility to alkylating chemotherapeutic agents and carcinogens. Using a newly developed flow cytometry assay with the monoclonal anti-AGT antibody, mT3.1, we compared AGT expression in single-cell suspensions with standard biochemical and Western blot assays to validate the fluorescence-activated cell sorting (FACS) method and develop potential applications. From Chinese hamster ovary cells (CHO) transfected with human O6-methylguanine-DNA methyl-transferase cDNA, 6 CHO-O6-methylguanine-DNA methyl-transferase clones were isolated that expressed 0.3 to 64 fmol/microgram DNA (by biochemical assay) of human AGT. FACS yielded a linear relationship between mean fluorescence intensity and both AGT activity by biochemical assay and AGT protein by Western blot. Using this standard curve, FACS-analyzed AGT protein content in human peripheral blood mononuclear cells (PBMCs) from normal donors ranged from 6.1 to 12.8 fmol/microgram DNA, similar to those obtained by biochemical assay and Western blot. This suggests that the level of immunoreactive protein appears to be an accurate predictor of AGT activity in the steady state. FACS-AGT in PBMCs from normal donors had a low index of heterogeneity within the sample. In contrast, by FACS-AGT analysis of human bone marrow samples and granulocyte-colony-stimulating factor-mobilized PBMCs, AGT was lower and had an 8-fold higher index of heterogeneity than observed in PBMCs from normal donors. After treatment with O6-benzylguanine (O6-bG), Western and FACS-AGT detected significant levels of AGT protein for up to 24 h, whereas biochemical assay showed AGT activity less than 5% of the basal level. Because only the biochemical assay accurately measures net AGT activity, the AGT-FACS assay will not be useful in clinical trials to assess the efficacy of O6-bG or other AGT inhibitors. Thus, AGT-FACS can rapidly assess the heterogeneity of steady-state AGT in single-cell suspensions and may be useful for assay in lymphocytes, bone marrow cells, leukemic myeloma plasma cells, or cells transfected with the AGT gene; Western blot analysis is better for small samples such as tumor biopsies, whereas biochemical assay is best able to measure enzyme activity and its inactivation by O6-bG or other agents.