Holoprosencephaly (HPE) is a complex structural brain anomaly that results from incomplete cleavage of the forebrain. The prevalence of HPE at birth is low, and risk factors have been difficult to identify. Using data from a large multi-state population-based case-control study, we examined risk factors for non-syndromic HPE. Data from maternal telephone interviews were available for 74 infants with HPE and 5871 controls born between 1997 and 2004. Several characteristics and exposures were examined, including pregnancy history, medical history, maternal diet and use of nutritional supplements, medications, tobacco, alcohol, and illegal substances. We used chi(2)-tests and logistic regression (excluding women with pre-existing diabetes) to examine associations with HPE. Except for diet (year before pregnancy) and sexually transmitted infections (STIs) (throughout pregnancy), most exposures were examined for the time period from the month before to the third month of pregnancy. HPE was found to be associated with pre-existing diabetes (chi(2) = 6.0; P = 0.01), aspirin use [adjusted odds ratio (aOR) = 3.4; 95% confidence interval (CI) 1.6-6.9], lower education level (aOR = 2.5; 95%CI 1.1-5.6), and use of assisted reproductive technologies (ART) (crude OR = 4.2; 95%CI 1.3-13.7). Consistent maternal folic acid use appeared to be protective (aOR = 0.4; 95%CI 0.2-1.0), but the association was of borderline statistical significance. While some of these findings support previous observations, other potential risk factors identified warrant further study.