Histamine N-methyltransferase (HNMT) catalyzes the N(tau)-methylation of histamine. The level of HNMT activity in human red blood cells is controlled by a common genetic polymorphism. We previously cloned and expressed a cDNA for human kidney HNMT, and we have now determined the structural organization of the human HNMT gene as a step toward studies of the genetic regulation of levels of HNMT activity in human tissue. Structural characterization of the HNMT gene was performed by use of a polymerase chain reaction (PCR)-based strategy. The gene was approximately 34 kb in length and contained 6 exons. All exon-intron splice junction sequences conformed to the "GT-AG" rule. The longest transcript isolated after 5'-rapid amplification of cDNA ends indicated that transcription initiation occurred 252 nucleotides 5'-upstream from the cDNA translation initiation codon. HNMT mapped to human chromosome 2. Structural characterization of the gene for HNMT will make it possible to study molecular genetic mechanisms involved in the regulation of this important enzyme in humans.