Hydroxyindole O-methyltransferase (EC 2.1.1.4) is the enzyme that catalyses the synthesis of melatonin in the pineal gland and in the retina. Polyadenylated RNA from chicken pineal glands was used to prepare a cDNA library in lambda gt11. The library was screened with an antiserum directed against chicken hydroxyindole O-methyltransferase, and one cDNA clone was isolated. The fusion protein expressed by phage lysogens was identified on Western blots as a 165 kDA immunoreactive protein (beta-galactosidase, 110 kDa; hydroxyindole O-methyltransferase, 38 kDa). The fusion protein exhibited hydroxyindole O-methyltransferase activity. Its Km values for N-acetyl-5-hydroxytryptamine and S-adenosylmethionine were 5 times those of the natural enzyme. The intrinsic activity of the fusion protein was approx. 0.25% that of the natural enzyme. The cDNA consisted of 1436 nucleotides, including a 1038-nucleotide sequence encoding a full-length 346-amino-acid hydroxyindole O-methyltransferase. Comparison with bovine hydroxyindole O-methyltransferase [Ishida, Obinata & Deguchi (1987) J. Biol. Chem. 262, 2895-2899] revealed 52% identity in nucleotide sequences and 44% identity in peptide sequences. Northern-blot analysis revealed the presence of hydroxyindole O-methyltransferase mRNA transcripts in chicken pineal gland and retina, but not in the telencephalon.