BACKGROUND:PAX3 plays an important role in mammalian embryonic development. Known mutations in PAX3 are etiologically associated with Waardenburg syndrome and syndromic neural tube defects (NTDs). Mutations in the murine homologue, pax3, are responsible for the phenotype of splotch mice, in which nullizygotes are 100% penetrant for NTDs. METHODS: The study sample included 74 infants with spina bifida (cases) and 87 nonmalformed infant controls. The conserved paired-box domain as well as the upstream genomic region of PAX3 were subjected to resequencing and those identified SNPs were evaluated as haplotypes. The associations of haplotypes for selected gene regions and the risks of spina bifida were further studied. RESULTS: Nineteen SNPs were observed; 15 observed in controls had been submitted to the National Center for Biotechnology Information (NCBI) database with allele frequencies. The PAX3 gene variant T-1186C (rs16863657) and its related haplotype, TCTCCGCCC of nine SNPs, were found to be associated with an increased risk of spina bifida, with an OR of 3.5 (95% CI: 1.2–10.0) among Hispanic Whites. CONCLUSIONS: Our analyses indicated that PAX3 SNPs were not strong risk factors for human spina bifida. However, additional follow-up of the PAX3 gene variant T-1186C (rs16863657) and its related haplotype, TCTCCGCCC, may be important in other populations. Birth Defects Research (Part A), 2007. © 2006 Wiley-Liss, Inc.