Three novel human NAT2 alleles (NAT2*5D, NAT2*6D, and NAT2*14G) were identified and characterized in a yeast expression system. The common rapid (NAT2*4) and slow (NAT2*5B) acetylator human NAT2 alleles were also characterized for comparison. The novel recombinant NAT2 allozymes catalyzed both N- and O-acetyltransferase activities at levels comparable with NAT2 5B and significantly below NAT2 4, suggesting that they confer slow acetylation phenotype. In order to investigate the molecular mechanism of slow acetylation in the novel NAT2 alleles, we assessed mRNA and protein expression levels and protein stability. No differences were observed in NAT2 mRNA expression among the novel alleles, NAT2*4 and NAT2*5B. However NAT2 5B and NAT2 5D, but not NAT2 6D and NAT2 14G protein expression were significantly lower than NAT2 4. In contrast, NAT2 6D was slightly (3.4-fold) and NAT2 14G was substantially (29-fold) less stable than NAT2 4. These results suggest that the 341T --> C (Ile(114) --> Thr) common to the NAT2*5 cluster is sufficient for reduction in NAT2 protein expression, but that mechanisms for slow acetylator phenotype differ for NAT2 alleles that do not contain 341T --> C, such as the NAT2*6 and NAT2*14 clusters. Different mechanisms for slow acetylator phenotype in humans are consistent with multiple slow acetylator phenotypes.