The aim of this study was to investigate N‐acetyltransferase 2 (NAT2) genetic polymorphism and enzyme activity in Serbs, and to examine the influence of NAT2 genotype, sex, and smoking on the phenotype. Genotyping for 190C>T, 282C>T, 341T>C, 403C>G, 411T>A, 481C>T, 590G>A, 803A>G, and 857G>A in the NAT2 gene, was performed in 140 healthy Serbs. NAT2 activity was determined as AFMU/(AFMU + 1X + 1U) urinary ratio in 100 subjects using caffeine as a probe. The most frequent NAT2 haplotypes were NAT2*5B (38.2%), NAT2*6A (26.0%), and NAT2*4 (24.4%). The log‐transformed NAT2 activity indices exhibited trimodal distribution with 9%, 36%, and 55% of slow, intermediate, and rapid acetylators, respectively. Significant NAT2 genotype‐phenotype correlation was observed (P < .0001). The frequency of NAT1*10 and NAT1*11 were 27.5% and 6.9%, respectively. There was no significant influence of sex or cigarette smoking on NAT2 enzyme activity. Eight subjects displayed rapid NAT2 acetylators phenotype despite being homozygous for NAT2 slow alleles, and NAT1 fast acetylators genotype (NAT1*10 and NAT1*11) had no implication. In contrast to other white populations described hitherto, rapid acetylator is the predominant NAT2 phenotype in Serbs. NAT2 genotype, but not sex and cigarette smoking, influence enzyme activity. NAT1 fast acetylators genotypes do not contribute for NAT2 genotype‐phenotype discordance.