The nucleotide sequence of the mRNA for malic enzyme ((S)-malate NADP+ oxidoreductase (oxaloacetate-decarboxylating, EC 22.214.171.124) from rat liver was determined from three overlapping cDNA clones. Together, these clones contain 2078 nucleotides complementary to rat liver malic enzyme mRNA. The single open reading frame of 1761 nucleotides codes for a 585-amino acid polypeptide with a calculated molecular mass of about 65,460 daltons. The cloned cDNAs contain the complete 3'-noncoding region of 301 nucleotides for the major mRNA species of rat liver and 16 nucleotides of the 5'-noncoding region. Amino acid sequences of seven tryptic peptides (67 amino acids) from the purified protein are distributed through the single open reading frame and show excellent correspondence with the translated nucleotide sequence. The putative NADP-binding site for malic enzyme was identified by amino acid sequence homology with the NADP-binding site of the enoyl reductase domain of fatty acid synthetase.