not-yet-known not-yet-known not-yet-known unknown The analysis of environmental DNA (eDNA) has become a non-invasive, cost-efficient and universal biomonitoring tool, widely applied across the globe. Most eDNA research focuses on aquatic organisms in freshwater and marine environments. eDNA shedding rates are key to interpretating eDNA-based results, such as for abundance estimations or detection probabilities. Shedding rates have been estimated for several species and life stages, however virtually all of them are aquatic. As eDNA-based biomonitoring expands to terrestrial systems, waterborne eDNA from freshwater is increasingly used to assess species presence of terrestrial mammals. While interacting with the water, terrestrial mammals deposit their DNA into the water body, with the amount deposited presumably heavily depending on the interaction type. Here we quantify eDNA shedding rates from domestic dogs during various interactions with water bodies, including ”passing by”, ”drinking”, ”crossing through”, ”standing still” and ”defecating.” “Crossing through” and “defecating” had the highest DNA shedding rates (both approx. 4x107 pg/h/ind). Since scat largely remains in the water once deposited, and the active movement of terrestrial mammals through the water is brief, we conclude that defecation is the major source of terrestrial mammal DNA in surface waters where direct defecation into the water is possible. This has important implications for interpretations of eDNA-based data from such water bodies. We also highlight the patchiness of eDNA particle distribution in such systems, which needs to be accounted for when designing eDNA sampling schemes.