Thyroid hormones substantially influence central nervous system development during gestation. We hypothesized that perturbations of early thyroid profiles may contribute to the development of autism spectrum disorders (ASD). Thyroid pathways could provide a mechanism by which environmental factors that affect the thyroid system may impact autism occurrence or phenotypic expression. We investigated whether thyroxine (T4) levels at birth are associated with subsequent ASD, using two existing California study groups in multivariate analysis. One study group included children born in the San Francisco Bay Area in 1994, with cases identified through the California Department of Developmental Services (DDS) and/or the Kaiser Permanente Medical Care Program of Northern California (244 cases, 266 controls); the other included children born in California in 1995, with cases identified through DDS (310 cases, 518 controls). Matched controls were selected from birth certificate records. This exploratory analysis suggested that infants with very low T4 (<3rd percentile) may have higher ASD risk, although results reached statistical significance only for the 1995 study group (1995: OR = 2.74 (95% CI 1.30–5.75; 1994: OR = 1.71 (95% CI 0.57–5.19). A variety of alternate analyses were conducted with available data, without further resolution of the difference between the two study groups. The results of our study indicate that further studies are warranted to investigate whether thyroid hormone perturbations play a role in the development of ASD by evaluating additional potential confounders and genotype or phenotype in larger studies. Autism Res2011,4:456–463. © 2011 International Society for Autism Research, Wiley Periodicals, Inc.