The gene for LSP1 is a lymphocyte-specific gene previously isolated by us using a subtractive hybridization technique. LSP1 mRNA is found in normal and transformed B lymphocytes and in normal T lymphocytes but not in transformed T lymphocytes. To study the expression of the mouse LSP1 protein, we prepared a polyclonal antiserum specific for the LSP1 protein. Here we report that the gene for LSP1 was expressed in transformed B-lymphoma cell lines and in normal mouse thymocytes as a protein doublet with apparent molecular masses of 52 and 50.5 kilodaltons when analyzed on a sodium dodecyl sulfate-10% polyacrylamide gel. BW5147 cells transfected with an LSP1 cDNA clone expressed only the 52-kilodalton protein. No LSP1 protein was expressed in nine T-lymphoma cell lines tested. Immunofluorescence studies of intact and permeabilized cells and subcellular fractionation experiments showed that the LSP1 protein was associated with the cytoplasmic side of the plasma membrane in transformed B-lymphoma cell lines and in normal thymocytes. Using a simple filter-binding assay, we showed that recombinant LSP1 protein was Ca2+ binding, as predicted on the basis of its deduced amino acid sequence. On the basis of the particular expression pattern, the subcellular localization, and the Ca2+-binding property of the LSP1 protein, we hypothesize that the LSP1 protein is a lymphocyte-specific component of a signal transduction pathway involved in the regulation of lymphocyte growth.