The prevalence and distribution of seven point mutations at the coding region of the highly polymorphic NAT2 gene were studied in 1008 chromosomes from healthy Spanish subjects. Most of the genes studied (78.4%) had one or more mutations, distributed in seventeen allelic variants of the NAT2 gene. Three alleles were present at high frequencies, namely NAT2*5B (41.6%), NAT2*6A (23.6%) and NAT2*4 (21.6%). The frequencies for the rest of alleles were: NAT2*12A (2.5%), NAT2*6B (2.0%), NAT2*13 (1.9%), NAT2*5A (1.5%), NAT2*7B (1.2%), NAT2*12C (1.0%), NAT2*5C (0.8%), NAT2*14C (0.8%), NAT2*14A (0.6%), NAT2*5D (0.3%), NAT2*12B (0.2%), and NAT2*14D (0.1%). In addition, we identified two new allelic variants with mutations at 191A + 341C + 803G (0.1%) and 282T + 590A + 803G (0.3%) which to our knowledge are described here for the first time. No other combination of mutations was identified, including the previously described allelic variants NAT2*14B, NAT2*14E, NAT2*5E and NAT2*7A. The phenotype predictive capacity of simplified PCR tests including analyses for mutations at 341C and 590A, and more sophisticated tests analysing seven mutations revealed that, in the population studied, the analysis of these two mutations is enough to predict as rapid acetylators over 99.5% subjects with two rapid genes, and about 94% subjects with one rapid gene. Given a prevalence of poor acetylators of about 55% subjects, the simplified analysis would predict the phenotype in about 97.5% subjects.