Glycogen synthase kinase-3 (GSK-3) is a protein-serine kinase implicated in the hormonal control of several regulatory proteins including glycogen synthase and the transcription factor c-jun. Two classes of rat brain cDNA for this enzyme have been isolated termed GSK-3 alpha and GSK-3 beta. The alpha-type encodes a 51 kd polypeptide, the sequence of which includes all of the tryptic peptides determined by protein sequence analysis of purified skeletal muscle GSK-3. The novel beta-type cDNA has the potential to encode a 47 kd protein with 85% amino acid identity to GSK-3 alpha. The two types of cDNA are the products of distinct genes as determined by genomic organization and nucleic acid sequence analysis. Both alpha and beta clones exhibit kinase activity when expressed in COS-1 cells and type-specific antibodies to GSK-3 alpha and beta detect proteins of 51 and 47 kd, respectively, in a variety of rat tissue extracts, with highest levels of both in brain. Partial purification of GSK-3 activity from bovine brain results in the isolation of active alpha and beta proteins. The physiological importance of these two proteins in cellular signal transduction is discussed.