Functions and phenotypes change in macrophages (Mφ) during tumor growth. Although analyzing functional and phenotypic changes are important in understanding the mechanism of tumor-induced immunosuppression, it is necessary to look beneath the surface and expose the mechanisms behind these changes. Flow cytometrically isolated Mac-1⁺, -2⁺, or -3⁺ Mφ showed that although both normal host and tumor-bearing host (TBH) Mac-2⁺ Mφ were the primary source of prostaglandin E2, no specific TBH suppressor Mφ could be identified. To determine if normal host and TBH Mφ respond to in vitro activating agents differently, normal host and TBH Mø, were treated with lipopolysaccharide. Functional, phenotypic, and molecular changes were observed in the lipopolysaccharide-treated M4J. Three- and 24-h lipopolysaccharide treatment reduced TBH Mφ-mediated suppression, while only 24-h lipopolysaccharide treatment reduced it in normal host Mφ. Prolonged adherence, which induces Mφ differentiation, increases the number of Mac-2⁺ TBH Mφ. Tumor growth causes an increase of Mφ in the S and G₂/M phases of the cell cycle. Lipopolysaccharide and adherence increase the number of normal host Mφ in Sand G₂/M; however, these same treatments reduce the number of TBH Mφ in these same phases. Earlier work showed an increase in the number of TBH Mφ that did not express class II major histocompatibility complex molecules and that there was an increase in the suppression mediated by these Mφ. TBH Mφ have a decreased response to interferon-γ-induced class II mRNA expression and a decrease in its mRNA stability. TBH Mφ have an increase in lipopolysaccharide- and prostaglandin E₂-mediated suppression of class II mRNA. Tumor necrosis factor-α can induce class II mRNA expression in TBH Mφ, but suppresses it in normal host Mφ. The effects of tumor necrosis factor-α on class II mRNA is due, in part, to the maturation stage of the Md. To examine further mechanisms that regulate Mφ maturation, intracellular Mac-2 expression was examined. The expression of nuclear Mac-2 increases during tumor growth and after 24-h adherence. This increase in Mac-2 protein parallels the increase in Mac-2 mRNA expression. Because there is a change in TBH Mφ cell-cycle kinetics and maturation, proto-oncogene expression was examined in normal host and TBH Mφ. The proto-oncogenes c-myb, c-myc, c-fos, and c-fms are constitutively expressed in TBH Mφ, while normal host M@ express c-fos and c-fms but at lower levels. Adherence suppresses the expression of the proto-oncogenes c-myb, c-myc, and c-fms in TBH Mφ, while inducing c-fos and c-fms in the normal host. Lipopolysaccharide induces c-myc, c-fos and c-fms in both normal and TBH Mφ, but suppresses c-myb expression in TBH Mφ. The results suggest that tumor growth causes a shift in Mφ maturity, and that this shift is responsible for alterations in Mφ function and phenotype. It is possible, however, to activate Mφ by lipopolysaccharide in the absence of triggering differentiation, and to trigger differentiation by adhesion without activating Mφ. TBH Mφ are more suppressive than normal host Mφ, but TBH Mφ can respond to activating signals.