Guanine nucleotide-binding regulatory proteins, or G proteins, mediate the interaction of agonist receptors on the platelet surface with phospholipase C and adenylyl cyclase. To better understand this process, we have used several approaches to identify which G proteins are present in platelets, normal human megakaryocytes, and human erythroleukemia (HEL) cells, a leukemic cell line with megakaryocytic features. Because platelet and HEL cell responses to thrombin are inhibited by pertussis toxin, we have focused upon the members of the G_i family, whose α subunits can be ADP-ribosylated by that toxin. Western blots with antisera specific for G_iα demonstrated the presence in both platelets and HEL cells of the three best-described forms of this protein: G_iα1, G_iα2, and G_iα3. Based upon immunoprecipitation studies with [³⁵S]-methionine-labeled HEL cells, their relative abundance appears to be G_iα2 > G_iα3 > G_iα1. A HEL cell cDNA library screened with the G_iα antisera produced clones encoding G_iα2 and G_iα3 that had sequences similar to those reported from other sources. G_iα-specific probes created from these cDNA clones confirmed the presence of mRNA encoding G_iα2 and G_iα3 in both platelets (by Northern blotting) and megakaryocytes (by in situ hybridization). Thus the pertussis toxin substrates that have previously been detected in platelets and HEL cells are shown to be members of the G_iα family, all of which are candidates for interaction with receptors for thrombin and other agonists.