Mononuclear phagocytes are functionally heterogeneous. To study the relationship of the heterogeneous populations of macrophages from the lung, alveolar macrophages from Syrian hamsters that had been immunized and rechallenged intratracheally with Mycobacterium bovis (Strain BCG) were separated by discontinuous albumin gradient centrifugation into 5 subpopulations designated A, B, C, D, and E. An activated alveolar macrophage subpopulation (defined by the ability to destroy tumor target cells) was enriched only in fraction D. Cells in fraction D destroyed 40% of the tumor cells, whereas unseparated alveolar macrophages destroyed 29%. Fractions A, B, C, and E destroyed less than 15% of the tumor cells. The subpopulations were functionally heterogeneous with regard to chemotactic responsiveness, Fc receptor activity, and phagocytic activity. Incubation of the subpopulations with a lymphocyte supernatant from spleen cells stimulated with concanavalin A enhanced the cytotoxic activity of fraction D and activated cells in fractions C and E to destroy tumor cells. Neither resident alveolar macrophages nor any of the subpopulations destroyed tumor cells. Only resident fraction D cells killed tumor cells when incubated with lymphokine containing supernatant fluids. The results are consistent with the hypothesis that some subpopulations of alveolar macrophages may be related and exist as a continuum of differentiation.