Hydroxyindole-O-methyltransferase (HIOMT) catalyzes the last step in the synthesis of the pineal hormone melatonin. In this study, an HIOMT clone was isolated from a human pineal cDNA library using synthetic oligonucleotide probes based on the bovine HIOMT sequence. The human sequence is unusual because it contains a 3' fragment (84 bp) of the LINE-1 sequence, a highly repetitive sequence in the human genome and the genome of some primates and rodents. Exclusive of this LINE-1 fragment, the human HIOMT clone is 75% and 63% homologous to bovine and avian HIOMT sequences, respectively. The deduced amino acid sequence of the human cDNA clone encodes a 41.6-kD protein. In addition, the sequence is 70% and 57% identical and 81% and 73% similar to bovine and avian HIOMT, respectively. In agreement with the results of earlier studies, it was found that vertebrate HIOMT amino acid sequences are not homologous to any other vertebrate proteins, including several methyltransferases. However, HIOMT exhibits homology with a plant O-methyltransferase and an internal 120-amino-acid region is approximately 35% identical to a region of four bacterial O-methyltransferases. The results of PCR and Southern blot analysis indicate that three species of HIOMT mRNA are typically present in the human pineal gland, only one of which contains the LINE-1 fragment. An antiserum was raised against a mixture of three synthetic peptides, corresponding to three regions of the deduced amino acid sequence of human HIOMT. This antiserum detected a single immunoreactive protein in Western blot analysis of human pineal glands. The size of the protein (approximately 42 kD) is identical to that predicted from the HIOMT clone, including the LINE-1 fragment. The human HIOMT sequence should be useful in further studies of this enzyme and will also be of special importance in evaluating the functional significance of the inclusion of a fragment of the LINE-1 in an mRNA.