We examined prenatal screening markers and offspring autism spectrum disorder (ASD) using California statewide data on singleton births in 1996 and 2002. Second trimester levels of unconjugated estriol (uE3), human chorionic gonadotropin (hCG), and maternal serum alpha-fetoprotein (MSAFP) were compared between mothers of children with ASD (n = 2586) and of non-cases (n = 600,103). Adjusted odds ratios (AOR) were calculated by logistic regression. Lower uE3 (AOR for < 10th percentile vs. 25th-74th percentiles = 1.21, 95% CI 1.06-1.37), and higher MSAFP (AOR = 1.21, 95% CI 1.07-1.37 for > 90th percentile) were significantly associated with ASD. A U-shaped relationship was seen for hCG (AOR = 1.16, 95% CI 1.02-1.32 for < 10th percentile; AOR = 1.19, 95% CI 1.05-1.36 for > 90th percentile). Our results further support prenatal hormone involvement in ASD risk.