Clinical use of diagnostic ultrasound imaging during pregnancy has a long history of safety and diagnostic utility, as supported by numerous human case reports and epidemiological studies. However, there exist in vivo studies linking large but clinically relevant doses of ultrasound applied to mouse fetuses in utero to altered learning, memory, and neuroanatomy of those mice. Also, there exists a well-documented significant increase in the likelihood of non-right-handedness in boys exposed to diagnostic ultrasound in utero, potentially relevant given the increased prevalence of autism in males, and reports of excess non-right-handedness in this population. Motivated by these observations, we applied 30 minutes of diagnostic ultrasound to pregnant mice at embryonic day 14.5 and assayed the social behavior of their male pups 3 weeks after their birth. The ultrasound-exposed pups were significantly (P < 0.01) less interested in social interaction than sham-exposed pups in a three-chamber sociability test. In addition, they demonstrated significantly (P < 0.05) more activity relative to the sham-exposed pups, but only in the presence of an unfamiliar mouse. These results suggest that fetal exposure to diagnostic ultrasound applied in utero can alter typical social behaviors in young mice that may be relevant for autism. There exist meaningful differences between the exposure of diagnostic ultrasound to mice versus humans that require further exploration before this work can usefully inform clinical practice. Future work should address these differences as well as clarify the extent, mechanisms, and functional effects of diagnostic ultrasound's interaction with the developing brain. Autism Res 2014, 7: 295–304. © 2013 International Society for Autism Research, Wiley Periodicals, Inc.