CD19, a B cell-specific transmembrane protein, is essential for murine B-1 cell development and T cell-dependent B cell immune responses. Whereas signaling by the human B cell Ag receptor can be modulated by CD19, less is known about the biochemical properties of murine CD19. We have used a novel rat mAb specific for murine CD19 to study the biochemical properties of the murine protein. We demonstrate that murine CD19 shares with human CD19 an association with complement receptor CD21 and CD81, tyrosine phosphorylation, binding of phosphatidylinositol-3 kinase, and synergistic signaling with membrane IgM. Murine CD19 is shown also to enhance signaling through the micro-surrogate light chain complex of primary pre-B cells. We found that although expressed in the earliest B cell precursors, CD19 ligation does not activate Ca2+ mobilization until the pre-B cell stage of development. In mature B cells, CD19 cross-linking activates Ca2+ flux in B-2 cells but not in B-1 cells, although it can synergize with surface IgM in both B-1 and B-2 cells. These biochemical properties of CD19 will be important for understanding its function in B cell development and the humoral immune response.