Prenatal testosterone (PT) effects have been proposed to increase systemizing (the drive to understand lawful input–output relationships), to decrease empathizing (the drive to understand others), and to cause autism via hypermasculinization of the brain. Digit ratio 2D:4D is a putative marker of PT effects in humans. An online study (n = 1896) into the relationship between the Reading the Mind in the Eyes Test (a widely used measure of empathizing) and self-measured 2D:4D in a nonclinical sample is reported. No evidence for a link between empathizing and 2D:4D in either females or males emerged. Further, three meta-analyses are presented that look into the relationships of 2D:4D with autism spectrum disorder (ASD), systemizing, and empathizing. 2D:4D was substantially lower (more masculine) in ASD-affected individuals than in normal controls (d = −0.58, P < 0.001). However, 2D:4D was found to be virtually unrelated to systemizing and empathizing in normal adults. The results support the idea that high PT is a risk factor for autism, but they challenge the view that PT substantially contributes to sex differences in systemizing and empathizing. Possibly, this pattern reflects an interaction effect, whereby PT drives ASD characteristic changes only in brains with a specific damage. Autism Res 2012, 5: 221–230. © 2012 International Society for Autism Research, Wiley Periodicals, Inc.