Concanavalin A is a potent stimulant of in vitro blast transformation and of DNA synthesis by murine lymphoid cells. In contradistinction to phytohemagglutin which stimulates thymocytes minimally, concanavalin A causes marked responses on the part of both thymus cells and spleen cells. Both of these stimulants lead to meager responses on the part of bone marrow cells. In the spleen and the bone marrow, the cells responsive to both agents appear to be thymus derived or thymus dependent. Thus, treatment of bone marrow cells with complement (C) and iso-antisera directed against the thymus dependent differentiation antigen ϑ abolishes the Con A and phytohemagglutinin (PHA) responsiveness of these cells. Spleen cells from adult mice which have been thymectomized, irradiated and reconstituted with bone marrow cells exhibit depressed responses to Con A and are unresponsive to PHA. Treatment of these cells with anti-ϑ antisera and C abolishes their residual Con A responsiveness. Despite differences in the reactivity in various lymphoid cell populations to Con A and to PHA, similar amounts of radioiodinated mitogen can be bound to the surface of these cells. Thus, the number of Con A molecules which can bind to the highly responsive spleen and thymus cell populations is similar (∼10 × 106) to that which binds to the weakly reactive bone marrow cells. Spleen cells from thymus-deprived mice bind similar amounts of Con A and PHA as do spleen cells from intact mice. Among given lymphoid cell populations, a heterogeneity in Con A binding can be demonstrated either by fractionation of the population on Con A-polyacrylamide affinity columns or by direct light and electron microscopic visualization using covalent complexes of Con A with horseradish peroxidase or with ferritin. An estimate of the number of Con A molecules which are required to bind to a cell surface to yield maximal stimulation was obtained by presensitizing thymus and spleen cells at 4°C with varying concentrations of Con A. Binding of approximately 1.5 to 2.5 × 106 molecules to these cells yielded optimal stimulation. These results are discussed in terms of heterogeneity of mouse lymphoid cell populations and of the complex requirements for cellular stimulation by mitogen.