As a tumor grows, changes occur in the function of macrophages (M phi). This is concomitant with changes in their phenotype. Flow cytometric analysis of monoclonal antibody (mAb)-labeled thioglycollate-elicited peritoneal, and resident splenic, M phi showed a tumor-induced shift of Mac-1, -2, -3, and Ia antigen expression. During tumor growth, the percentage of peritoneal Mac-2+, -3+, and Ia+ M phi decreased significantly (22%, 14%, and 58%, respectively), while Mac-1+ M phi remained unchanged. By analyzing the data on two-dimensional histograms and comparing the sizes of M phi to cell-surface antigen expression, we identified distinct subpopulations of peritoneal M phi. Three distinct size versus antigen expression M phi subpopulations were detected by flow cytometry and consisted of 10-16, 17-22, and 23-27 microns for the small-, medium-, and large-sized populations, respectively. Large-sized Mac-1+ and -2+ M phi decreased (37% and 38%), while large-sized Mac-3+ M phi did not decrease during tumor growth. Medium-sized Mac-3+ M phi decreased 33% during tumor growth, while no differences could be seen in medium-sized Mac-1+ or -2+ M phi. Concomitant with the decrease in large-sized Mac-1+ M phi was an increase in small-sized Mac-1+ M phi. Peritoneal Ia+ M phi were mostly small-sized (4-7-fold increase over the medium-sized and none in the large-sized population). M phi Ia antigen expression was nearly absent in the 21-day tumor-bearing host, with less than 4% of the cells labeling positive (a 73% drop from normal host M phi). In splenic M phi, the percentage of Mac-1+ M phi significantly increased (90%) during tumor growth, while Mac-2+ and -3+ M phi showed a smaller, but still significant, increase (48% and 40%, respectively). Additionally, splenic Ia+ M phi significantly decreased (29%) during tumor growth. More important than the decreased cell numbers was the significant decrease in Ia antigen expression per cell. Unlike the peritoneal M phi, the splenic M phi did not show distinct size versus antigen expression subpopulations, although there was an overall difference in M phi size between normal and TBH. These data suggested that M phi from different anatomical sites are phenotypically different and tumor growth mediates phenotypic alterations in peritoneal and splenic M phi populations. This may be the source of tumor-induced dysfunction of M phi-mediated immune activity.