Previous studies have demonstrated differences in immunologic function among density-fractionated alveolar macrophages. The present study was undertaken to correlate these functional differences among alveolar macrophage density fractions with parameters of macrophage activation. Alveolar macrophages were lavaged from normal rats and separated into 5 density fractions by density gradient centrifugation. Cells from each density fraction were analyzed for parameters of macrophage activation: in vitro cytotoxic function directed against cultured neoplastic cells, ectoenzyme activities, and surface expression of Ia-like determinants. Lavaged alveolar macrophages with in vitro cytotoxic function were concentrated among cells from higher density fractions, and cytotoxic function among the density fractions correlated inversely with ectoenzyme activities. Although the percentage of Ia-positive cells varied among the density fractions, surface expression of Ia-like determinants did not correlate positively with in vitro cytotoxic function. The results show that there is a variable distribution of cytotoxic function among alveolar macrophage density fractions that correlates with enzymatic markers of activation. Thus, we conclude that differential macrophage activation is a potential determinant of the functional heterogeneity observed in rat alveolar macrophages.