A complementary DNA (cDNA) encoding ornithine decarboxylase was isolated from a human liver cDNA library, and the nucleotide sequence coding for the entire enzyme was determined. The 1825-nucleotide-long cDNA contained an open reading frame of 1383 nucleotides, 87 nucleotides 5' from the first methionine codon, 346 nucleotides in the 3'-noncoding region, and a poly(A) tail of nine bases. Primer extension studies indicated that the 5'-noncoding region of the human ornithine decarboxylase mRNA was 335 nucleotides long. The amino acid sequence deduced from the open reading frame for a 461-residue polypeptide predicts a molecular weight of 51.156 for the human enzyme and has about 90% homology with the amino acid sequence of the murine ornithine decarboxylase (44 differences among the 461 amino acids). The nucleotide sequences of the human and murine ornithine decarboxylase mRNAs share an 85% homology, even in their 3'-noncoding regions. In contrast to rodent tissues with two ornithine decarboxylase mRNAs, normal human tissues appear to express only a single mRNA species with a molecular size of 2.25 kb. Southern blotting of human leukocyte DNA from 20 individuals indicated that the ornithine decarboxylase gene belongs to a multigene family in man and showed restriction fragment length polymorphism when cleaved with Pst I, but not when cleaved with Pvu II, Msp I, Hinc II, or Bam HI.