As a result of alternative splicing, the fyn gene encodes two different tyrosine protein kinase isoforms. While one protein (p59fynB) is abundantly expressed in the brain, the alternative product (p59fynT) is contained only in cells of hemopoietic lineages, especially T lymphocytes. Sequence analyses have revealed that these two isoforms differ exclusively within a stretch of 52 amino acids which overlaps the end of the Src homology 2 (SH2) motif and the beginning of the catalytic domain. Consistent with the idea that FynT provides a specialized function in hemopoietic cells, we have previously shown that expression of activated FynT molecules, but not that of activated FynB polypeptides, enhanced the antigen responsiveness of a mouse T-cell line (BI-141) (D. Davidson, L. M. L. Chow, M. Fournel, and A. Veillette, J. Exp. Med. 175:1483-1492, 1992). In this study, we examined the basis for the distinct signalling capabilities of the two Fyn isoforms in T lymphocytes. Our biochemical analyses revealed that FynT is more adept than FynB at promoting antigen receptor-triggered calcium fluxes. This phenomenon likely contributes to the improved biological function of FynT during antigen stimulation, as the calcium ionophore ionomycin partially rescued the inability of FynB to enhance antigen-induced lymphokine secretion. To establish the structural basis for these observations, we also created and analyzed a series of chimeras of FynT and FynB. These studies demonstrated that the distinct catalytic domain of FynT, and not its altered SH2 motif, is responsible for the improved ability to augment antigen responsiveness. Similarly, this sequence enhances the ability to mobilize cytosolic calcium in response to antigen receptor stimulation. Taken together, these data show that the distinct biological impacts of FynT and FynB in T cells are related to limited structural differences in the amino-terminal portion of their catalytic domains and that they reflect, at least in part, the greater ability of FynT to mobilize cytoplasmic calcium.