Urban and industrial air pollution can cause elevated heritable mutation rates in birds and rodents. The relative importance of airborne particulate matter versus gas-phase substances in causing these genetic effects under ambient conditions has been unclear. Here we show that high-efficiency particulate-air (HEPA) filtration of ambient air significantly reduced heritable mutation rates at repetitive DNA loci in laboratory mice housed outdoors near a major highway and two integrated steel mills. These findings implicate exposure to airborne particulate matter as a principal factor contributing to elevated mutation rates in sentinel mice and add to accumulating evidence that air pollution may pose genetic risks to humans and wildlife.