Perchlorate is a persistent and mobile contaminant in the environment with both natural and anthropogenic sources. Stable isotope ratios of oxygen (δ18O, δ17O) and chlorine (δ37Cl) along with the abundance of the radioactive isotope 36Cl were used to trace perchlorate sources and behavior in the Laurentian Great Lakes. These lakes were selected for study as a likely repository of recent atmospheric perchlorate deposition. Perchlorate concentrations in the Great Lakes range from 0.05 to 0.13 μg per liter. δ37Cl values of perchlorate from the Great Lakes range from +3.0‰ (Lake Ontario) to +4.0‰ (Lake Superior), whereas δ18O values range from -4.1‰ (Lake Superior) to +4.0‰ (Lake Erie). Great Lakes perchlorate has mass-independent oxygen isotopic variations with positive δ17O values (+1.6‰ to +2.7‰) divided into two distinct groups: Lake Superior (+2.7‰) and the other four lakes (∼+1.7‰). The stable isotopic results indicate that perchlorate in the Great Lakes is dominantly of natural origin, having isotopic composition resembling that measured for indigenous perchlorate from preindustrial groundwaters of the western USA. The 36Cl/Cl ratio of perchlorate varies widely from 7.4 × 10-12 (Lake Ontario) to 6.7 × 10-11 (Lake Superior). These 36ClO4- abundances are consistent with an atmospheric origin of perchlorate in the Great Lakes. The relatively high 36ClO4- abundances in the larger lakes (Lakes Superior and Michigan) could be explained by the presence of 36Cl-enriched perchlorate deposited during the period of elevated atmospheric 36Cl activity following thermonuclear bomb tests in the Pacific Ocean.