Periconceptional maternal folic acid supplementation can prevent up to 70% of pregnancies affected with neural tube defects (NTDs), including spina bifida. This has focused attention on folate-related genes such as dihydrofolate reductase (DHFR) in a bid to identify the genetic factors that influence NTD risk through either the fetal or maternal genotype. We considered a novel intronic 19-bp deletion polymorphism and two polymorphisms within the 3′ untranslated region (721A > T and 829C > T) of the DHFR gene as candidates for NTD risk. We studied NTD cases (n = 283), mothers of cases (n = 280), fathers of cases (n = 279), and controls (n = 256). We did not find the DHFR 829C > T polymorphism to be variable within the Irish population. The 19-bp intron deletion and the 721A > T polymorphisms were found to be in linkage disequilibrium. In contrast to a previous study, the 19-bp intron deletion allele did show a significant protective effect in mothers of NTD cases when present in one (relative risk 0.59 [95%CI: 0.39-0.89], P = 0.01) or two copies (relative risk 0.52 [95%CI: 0.32-0.86], P = 0.01). Analysis of mRNA levels revealed a small increase in expression (∼1.5-fold) associated with the 19-bp intron deletion polymorphism, but this was not significant. In conclusion, the DHFR intron 19-bp deletion allele may be a protective NTD genetic factor by increasing DHFR mRNA levels in pregnant women. © 2007 Wiley-Liss, Inc.