RBL-2H3 rat basophilic leukemia cells were homogenized and fractionated. A fraction F3 obtained by differential centrifugation was 6-fold enriched in [3H]-inositol 1,4,5-trisphosphate (InsP3) binding activity, while the NADH-cytochrome c oxidoreductase and sulphatase-C activities were only 3.8- and 2.9-fold enriched, respectively. Furthermore, the three InsP3 receptor (InsP3R) isoforms, two sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) isoforms (2b and 3) as well as four Ca2+ binding proteins (calreticulin, calnexin, protein disulfide isomerase (PDI) and BiP), were present in this fraction. Fraction F3 was, therefore, further purified on a discontinuous sucrose density gradient, and the 3 resulting fractions were analyzed. The InsP3 binding sites were distributed over the gradient and did not co-migrate with the RNA. We examined the relative content of the three InsP3R isoforms, of both SERCA2b and 3, as well as that of the four Ca2+ binding proteins in fraction F3 and the sucrose density gradient fractions. InsP3R-1 and InsP3R-2 showed a similar distribution, with the highest level in the light and intermediate density fractions. InsP3R-3 distributed differently, with the highest level in the intermediate density fraction. Both SERCA isoforms distributed similarly to InsP3R-1 and InsP3R-2. SERCA3 was present at a very low level in the high density fraction. Calreticulin and BiP showed a pattern similar to that of InsP3R-1 and InsP3R-2 and the SERCAs. PDI was clearly enriched in the light density fraction while calnexin was broadly distributed. These results indicate a heterogeneous distribution of the three InsP3R isoforms, the two SERCA isoforms and the four Ca2+ binding proteins investigated. This heterogeneity may underlie specialization of the Ca2+ stores and the subsequent initiation of intracellular Ca2+ signals.