This study investigates hydrological controls on E. coli concentration and loading in two artificially drained agricultural watersheds (58 and 23 km(2)) of the U.S. Midwest. Stream E. coli concentrations are significantly (p < 0.02) lower at base flow than high flow; however, E. coli load is significantly higher at high flow than at low flow (p < 0.001). Although E. coli concentrations are not significantly higher (p = 0.253) in summer/fall (3269 MPN/100 mL) than in the winter/spring (2411 MPN/100 mL), E. coli load is significantly higher (p < 0.05) in winter/spring (346 MPN/day) than in summer/fall season (75 MPN/day). Correlation analysis indicates that discharge and precipitation are the best indicators of E. coli concentration and 7-d antecedent precipitation (7dP), the best indicator of E. coli loading in the watersheds studied regardless of flow conditions and location. However, E. coli concentration and loading best correlate to 7dP and turbidity at base flow. A spatial dependency is also observed at base flow with E. coli concentration and load correlating better to 7dP in the headwaters and to turbidity in the lower reaches of the watersheds studied. For high flow conditions, E. coli concentration and loading are poorly correlated to most variables, except stream water temperature and 7-d antecedent discharge. These results are consistent with those reported in the literature and suggest that, at least during base flow conditions, turbidity and 7dP may be usable in artificially drained landscapes of the Midwest to identify potential hot spots of E. coli contamination.