We isolated and sequenced a cDNA clone encoding the bovine "80- to 87-kDa" protein, a major cellular substrate for protein kinase C. An open reading frame of 1005 base pairs predicted a protein of 335 amino acids (Mr, 31,949). Despite this predicted size, the protein migrated on SDS/polyacrylamide gels with an apparent molecular weight of 80-87,000 after expression of the cDNA in cells lacking the protein. It was highly enriched in alanine (28.4 mol %), contained an amino-terminal myristoylation consensus sequence, and included a 25-residue basic domain containing the known protein kinase C phosphorylation sites. Two mRNA species (2.6 and 4.4 kilobases) were most highly expressed in brain, spinal cord, spleen, and lung, in parallel with the distribution of immuno-reactive protein. Genomic blot analysis indicated the likelihood of a single gene coding for this mRNA. We propose the name myristoylated alanine-rich C kinase substrate (MARCKS) for this protein.