The functional and morphologic aspects of immunologically relevant macrophage-lymphocyte interaction have been characterized in inbred guinea pigs. Antigen-dependent initiation of in vitro DNA synthesis in lymphocytes taken from animals previously immunized with that antigen has been shown to be macrophage dependent. Expression of this in vitro correlate of in vivo established cellular immunity requires that antigen is initially bound to macrophages antecedent to any interaction with the specific lymphocyte. In view of the failure of supernatants from macrophages briefly exposed to antigen to initiate DNA synthesis in immune lymphocytes and the critical density requirements for lymphocyte proliferation, it is reasonable to assume that a direct physical interaction occurs between the antigen-bearing macrophage and lymphocyte. Using an in vitro assay of lymphocyte binding to macrophage monolayers, we have characterized what appear to be sequential events in macrophage-lymphocyte interaction. The initial event is an antigen-independent interaction between macrophage and lymphocyte whose characteristics are rapid reversibility, species but not strain specificity, and dependence on macrophage metabolism. If macrophages bear antigen for which a given lymphocyte possesses a specific receptor, then a second nonreversible antigen-dependent stabilization of binding occurs. This latter step requires tht macrophage and lymphocyte be histocompatible. These observations parallel functional studies of the histocompatibility dependence of macrophage-associated antigen initiation of lymphocyte DNA synthesis in guinea pigs. A model sequence of the physical events correlating with functional macrophage-lymphocyte interaction is proposed.