A cDNA library was constructed from the pineal gland of rats injected with isoproterenol and screened with 32P-labeled cDNAs encoding arylamine N-acetyltransferases from rabbit and human liver. Two types of cDNAs for arylamine N-acetyltransferases (A-type and B-type) were isolated. Expression of the cDNAs in Chinese hamster ovary cells indicated that A-type N-acetyltransferase acetylates both arylamines and arylalkylamines, while the B-type enzyme acetylates only arylamines. Therefore, neither the A-type nor the B-type of enzyme seems to be the arylalkylamine N-acetyltransferase involved in melatonin synthesis in the pineal gland. Nucleotide sequence analysis revealed that both A-type and B-type cDNAs code for 290 amino acids, and that they showed 82.8% similarity in the coding region. However, the nucleotide sequence in the 5' non-coding region was identical in the A-type and B-type cDNAs. In addition, the 5' non-coding region contained another possible open reading frame for 79 amino acids. Data base research revealed that the complementary sequence of the 5' non-coding region has high similarity with the coding regions of cDNAs for high-mobility-group proteins (HMG) 1 and 2, which are thought to regulate mRNA transcription.