A new method for the analysis of the binding of monoclonal antibodies to cell surface tumor-associated antigens utilizes 1- to 2-day primary cultures of human colonic carcinomas, adenomas, and normal epithelial tissue. The antibodies are added to the live cells which form monolayer epithelial patches of several hundred cells on the surface of the Petri dish by migration in a continuous sheet from a small explant. These epithelial patches are then fixed with methanol and processed in situ using the indirect immunoperoxidase assay. Three monoclonal antibodies (MAbs) prepared against membrane-enriched fractions of human metastatic breast cancer were assayed. MAb B1.1 bound to each of 11 benign and each of 18 malignant colonic tumors tested. MAb B6.2 displayed similar reactivity, binding to each of 7 adenomas and each of 15 carcinomas assayed. Both MAbs also bound to normal colonic epithelial cells in both the live cell studies presented here and in earlier studies (D. Stramignoni et al., Int. J. Cancer, 31: 543-552, 1983). MAb B72.3 bound only to tumor cells and not to normal epithelial cells in the live cell assay. This epitope was rapidly lost in culture. B72.3 reactivity on each of two carcinomas was decreased 9- to 36-fold when primary culture continued for 5-6 days. B72.3 bound to each of 20 tumors (15 carcinomas, 5 adenomas) when the cells were cultured for 1 or 2 days but on only 2 of 8 tumors when the cells were cultured for 3 to 8 days. The B72.3 epitope was more strongly expressed on the live cells in the explant and on those monolayer cells directly adjacent to the explant than on the cells more towards the edges of the patch colony. This implied that the cell flattening which occurred when cells migrated from the explant may have played some role in antigen loss. A very similar fraction of primary cultured carcinoma and adenoma cells bound each MAb, indicating that these MAbs in live cell assay do not distinguish between benign, noninvasive colonic tumors and invasive carcinomas. The live cell assay was compared to the standard assay utilizing sectioned, fixed tumors. In parallel assays of eight tumors the fraction of cells reactive in the indirect immunoperoxidase assay was consistently higher on live cells for each of these MAbs than on fixed tissue. Due to this greater sensitivity the live cell assay was able to detect reactive cells in two cases which were scored as negative (less than 1% positive cells) in the fixed tissue assay.