A major mechanism of resistance to nitrosoureas is O6-alkylguanine-DNA-alkyltransferase. The alkyltransferase biochemical assay measures mean tissue activity but requires availability of fresh tissue and cannot assess tumor heterogeneity, an important component of tumor resistance to alkylating agents. We assessed the levels of alkyltransferase in human colon carcinoma and normal colon by biochemical assay, Western blot, conventional immunohistochemistry, and quantitative immunohistochemistry (using 5H7 and mT3.1 monoclonal IgGs) to correlate whole tissue levels with cell-specific expression. Alkyltransferase activity was 18.0 +/- 4.6 fmol/microgram DNA in normal colon and 15.0 +/- 6.5 fmol/microgram DNA in tumors. By Western blot estimates, alkyltransferase in normal colon was 14.8 +/- 4.2 fmol/microgram DNA and in tumors was 16.2 +/- 7.8 fmol/microgram DNA. Alkyltransferase estimates by biochemical and Western blots were correlated strongly (P < 0.0001). Conventional immunohistochemistry demonstrated that alkyltransferase was predominantly nuclear and in normal colon was concentrated in glandular epithelial mucosal cells close to the lumen, whereas in tumors, expression was heterogenous but localized to malignant epithelial cells. Two parameters of quantitative immunohistochemistry, integrated gray and mean gray, were correlated strongly with each other (P < 0.002) and with biochemical and Western blot estimates (P = 0.004-0.04). Thus, quantitative immunohistochemical estimates of alkyltransferase in fixed tissues are a reasonable alternative to biochemical analysis and have an added advantage of identifying heterogeneity of alkyltransferase expression in tumors.