Abstract: Water-quality standards have been placed on fecal indicator organisms such as Escherichia coli in an attempt to limit the concentrations in water bodies. Cattle can be a significant source of bacteria to water systems, particularly when they are allowed direct access to streams. A flume study was conducted to quantify the effect and understand the transport of E. coli from directly deposited cattle manure. Five steady-state flows, ranging from 0.00683 to 0.0176 m3/s, were studied and loads from a single cowpie exceeded the U.S. Environmental Protection Agency’s recommended water-quality standards (235 CFU/100 ml) at each flow over the hour study period. Average E. coli concentrations ranged from 102 to 105 CFU/100 ml over the hour sampling period for all flows. High spatial variations in E. coli concentrations were often seen at each sampling time, with higher concentrations typically at the bottom of the flume. E. coli resuspension was initially greater at 0.5 min after deposition, for the lowest flow (105 CFU/m2/s); however, resuspension rates became similar over time, on the order of 103 CFU/m2/s. This study demonstrates that the concentrations of E. coli can vary over the water column, and therefore grab samples may inaccurately measure bacteria concentrations and loads in streams. In addition, resuspension rates were often high, so the incorporation of this process into water-quality models is important for bacteria prediction.