A complementary DNA (cDNA) has been isolated that encodes a second form of the beta subunit of signal-transducing guanine nucleotide-binding regulatory proteins (G proteins). The cDNA corresponds to a 1.8-kilobase mRNA, and nucleotide sequence analysis indicates that the encoded polypeptide consists of 340-amino acid residues with a Mr of 37,335. Although the deduced polypeptide is the same size as that reported previously for the beta subunit, 10% of the amino acid residues are different. Furthermore, the 5' and 3' non-translated regions of this cDNA show no significant homology with those reported previously for cDNAs that encode the beta subunit. These data refute prior conclusions that there is only one form of the G protein beta subunit.