Ice-nucleating particles (INPs) associated with fresh waters are a neglected, but integral component of the water cycle. Abundant INPs were identified from surface waters of both the Maumee River and Lake Erie with ice nucleus spectra spanning a temperature range from −3 to −15 °C. The majority of river INPs were submicron in size and attributed to biogenic macromolecules, inferred from the denaturation of ice-nucleation activity by heat. In a watershed dominated by row-crop agriculture, higher concentrations of INPs were found in river samples compared to lake samples. Further, ice-nucleating temperatures differed between river and lake samples, which indicated different populations of INPs. Seasonal analysis of INPs that were active at warmer temperatures (≥−10 °C; INP −10 ) showed their concentration to correlate with river discharge, suggesting a watershed origin of these INPs. A terrestrial origin for INPs in the Maumee River was further supported by a correspondence between the ice-nucleation signatures of river INPs and INPs derived from the soil fungus Mortierella alpina. Aerosols derived from turbulence features in the river carry INP −10 , although their potential influence on regional weather is unclear. INP −10 contained within aerosols generated from a weir spanning the river, ranged in concentration from 1 to 11 INP m −3 , which represented a fold-change of 3.2 over average INP −10 concentrations sampled from aerosols at control locations.