Hydroxyindole-O-methyltransferase (HIOMT) catalyzes the final step in melatonin synthesis. The nucleotide and deduced amino acid sequences of bovine HIOMT have been reported. Our laboratory recently isolated a cDNA clone encoding human HIOMT. Comparison of the human and bovine nucleotide sequences revealed several discrepancies which prevented perfect alignment and produced defined regions of virtually no homology in the deduced amino acid sequence. Consequently, we repeated sequence analysis of the original bovine HIOMT cDNA clone, the results of which are reported here. The revised nucleotide sequence includes 23 differences from the published sequence. This completely changes the deduced amino acid sequence in two regions, encompassing a total of 96 residues, or 28% of the protein. The revised deduced amino acid sequence predicts different post-translational modifications as compared to that of the original deduced sequence. This information will make it possible in future investigations of HIOMT to design improved polymerase chain reaction primers, peptides for the generation of antisera, and probes for various types of analysis and screening of libraries.