Commercially available concanavalin A binds Ca2+ with high apparent affinity. In order to dissociate concanavalin A stimulated Ca2+ uptake (defined as an increased association of 45Ca2+ with cells) in rat splenocytes and Ca2+ binding to cell-bound concanavalin A, conditions were developed to remove more than 75% of the bound concanavalin A. Under these conditions concanavalin A treated cells showed a considerable increase in 45Ca2+ uptake over control. The concanavalin A stimulated uptake of 45Ca2+ occurred within minutes, and required concentrations of concanavalin A which promoted [3H]thymidine uptake into these cells. Succinyl concanavalin A was less potent in promoting Ca2+ uptake than concanavalin A. Sodium periodate inhibited Ca2+ uptake at concentrations which promoted 3H-thymidine incorporation into splenocytes. It is concluded that concanavalin A promotes Ca2+ uptake which is not due to binding of 45Ca2+ to concanavalin A. Although the concanavalin A-promoted Ca2+ uptake occurs at lectin concentrations that cause lymphocyte proliferation as measured by 3H-thymidine incorporation, the role of Ca2+ in this event remains unclear.