The ability of the macrophage to express class II MHC gene products appears to arise from both T-dependent and T-independent mechanisms. One mechanism by which macrophages express Ia-antigens in the absence of T-lymphocytes is postulated to be controlled by differentiation. By using a liquid bone marrow culture system, we have studied both class I and class II surface expression and mRNA accumulation during macrophage differentiation in vitro. The results demonstrated that Ia expression increased until 7 days in culture and then slowly declined. In contrast, class I expression appeared to steadily increase throughout the differentiation period. Northern blot analysis of RNA isolated from bone marrow-derived macrophages (BMDM) at various periods during culture, using E alpha, A alpha, and class I cDNA probes, correlated well with the results of Ia and H-2K surface expression. Further analysis demonstrated that the expression of Ia-antigens on BMDM was not the result of T-helper lymphocytes. This was determined by demonstrating (1) that bone marrow cultures were devoid of mature T-lymphocytes, (2) the absence of interferon (IFN)-gamma transcripts in both adherent and nonadherent populations of bone marrow cells, and (3) that the addition of anti-IFN-gamma monoclonal antibody (mAb) to the bone cultures did not alter the percentage of Ia-positive BMDM. Moreover, the addition of anti-tumor necrosis factor-alpha mAb to the bone marrow cultures had no effect on Ia expression by BMDM. Taken together, these results allow us to conclude that Ia expression by BMDM is probably not mediated via exogenous signals but rather results from an intrinsically controlled process.