We have purified three 35-kDa calcium- and phospholipid-binding proteins from rat liver. These three calcimedins bind to phosphatidylserine in a calcium-dependent manner and have been termed 35 alpha, 35 beta, and 35 gamma based on their relative charge as determined by isoelectric focusing. Purification of the three 35-kDa calcimedins is achieved by phenyl-Sepharose, ion exchange, and gel filtration chromatography. Antibody was produced against the annexin consensus peptide, Lys-Ala-Met-Lys-Gly-Leu-Gly-Thr-Asp-Glu, which was derived from the sequence of several Ca2+/phospholipid-binding proteins including calpactin, lipocortin, endonexin II, 67-kDa calelectrin, lymphocyte 68-kDa protein, and protein II. Recognition of each 35-kDa calcimedin by anticonsensus sequence antibody places them in this protein family. Antibodies against each 35-kDa calcimedin were raised and purified by antigen-affinity chromatography. Each antibody is monospecific for the respective 35-kDa calcimedin. Immunological cross-reactivity defines 35 alpha, 35 beta, and 35 gamma as lipocortins III, IV, and V, respectively. Surveys by immunoblot analysis using these monospecific antibodies demonstrate a markedly different tissue expression pattern for each 35-kDa calcimedin. Furthermore, the levels of 35 alpha, 35 beta, and 35 gamma are differentially regulated in maturing rat ovary and uterus. Each calcimedin has been localized by indirect immunofluorescence within specific cell types. These results support the concept that mediation of the intracellular calcium signal can occur via multiple pathways through several related yet independent mediator proteins.