AbstractParaoxonase 1 (PON1) prevents oxidation of low‐density lipoproteins and inactivates toxic oxon derivatives of organophosphate pesticides (OPs). More than 250 SNPs have been previously identified in the PON1 gene, yet studies of PON1 genetic variation focus primarily on a few promoter SNPs (−108, −162) and coding SNPs (192, 55). We sequenced the PON1 gene in 30 subjects from a Mexican‐American birth cohort and identified 94 polymorphisms with minor allele frequencies >5%, including several novel variants (six SNPs, one insertion, and two deletions). Variants of the PON1 gene and three SNPs from PON2 and PON3 were genotyped in 700 children and mothers from the same cohort. PON1 phenotype was established using two substrate‐specific assays: arylesterase (AREase) and paraoxonase (POase). Twelve PON1 and two PON2 polymorphisms were significantly associated with AREase activity, and 37 polymorphisms with POase activity; however, only nine were not in strong linkage disequilibrium (LD) with either PON1−108 or PON1192 (r2 > 0.20), SNPs with known effects on PON1 quantity and substrate‐specific activity. Single tagSNPs PON155 and PON1192 accounted for similar ranges of AREase variation compared to haplotypes comprised of multiple SNPs within their haplotype blocks. However, PON155 explained 11–16% of POase activity, while six SNPs in the same haplotype block explained threefold more variance (36–56%). Although LD structure in the PON cluster seems similar between Mexicans and Caucasians, allele frequencies for many polymorphisms differed strikingly. Functional effects of PON genetic variation related to susceptibility to OPs and oxidative stress also differed by age and should be considered in protecting vulnerable subpopulations. Environ. Mol. Mutagen. 52:105–116, 2011. © 2010 Wiley‐Liss, Inc.