GTP-binding proteins of the Rab family were cloned from human platelets using RT-PCR. Clones corresponding to two novel Rab proteins, Rab31 and Rab32, and to Rab11A, which had not been detected in platelets previously, were isolated. The coding sequence of Rab31 (GenBank accession no. U59877) corresponded to a 194 amino-acid protein of 21.6 kDa. The Rab32 sequence was extended to 1000 nucleotides including 630 nucleotides of coding sequence (GenBank accession no. U59878) but the 5' coding sequence was only completed later by others (GenBank accession no. U71127). Human Rab32 cDNA encodes a 225 amino-acid protein of 25.0 kDa with the unusual GTP-binding sequence DIAGQE in place of DTAGQE. Northern blots for Rab31 and Rab32 identified 4.4 kb and 1.35 kb mRNA species, respectively, in some human tissues and in human erythroleukemia (HEL) cells. Rabbit polyclonal anti-peptide antibodies to Rab31, Rab32 and Rab11A detected platelet proteins of 22 kDa, 28 kDa and 26 kDa, respectively. Human platelets were highly enriched in Rab11A (0.85 microg x mg of platelet protein(-1)) and contained substantial amounts of Rab32 (0.11 microg x mg protein(-1)). Little Rab31 was present (0.005 microg x mg protein(-1)). All three Rab proteins were found in both granule and membrane fractions from platelets. In rat platelets, the 28-kDa Rab32 was replaced by a 52-kDa immunoreactive protein. Rab31 and Rab32, expressed as glutathione S-transferase (GST)-fusion proteins, did not bind [alpha-(32)P]GTP on nitrocellulose blots but did bind [(35)S]GTP[S] in a Mg(2+)-dependent manner. Binding of [(35)S]GTP[S] was optimal with 5 microm Mg(2+)(free) and was markedly inhibited by higher Mg(2+) concentrations in the case of GST-Rab31 but not GST-Rab32. Both proteins displayed low steady-state GTPase activities, which were not inhibited by mutations (Rab31(Q64L) and Rab32(Q85L)) that abolish the GTPase activities of most low-M(r) GTP-binding proteins.