Escherichia coli transported in surface runoff from dissolution of applied poultry litter is a major variable in assessing fecal contamination of streams. However, the relative magnitude of the E. coli concentration from a specific poultry litter application and relative to the time lag between litter application and rainfall are not completely understood. This research investigated E. coli transport in runoff on fourteen, 2 m by 2 m pastureland plots. Poultry litter was manually applied (4,942 kg/ha) in twelve plots followed by artificial rainfall with intensities equivalent to 2-yr and 5-yr storm events. Rainfall was applied in duplicate plots immediately after poultry litter application and 24 and 120 hr after litter application. Experiments were also conducted on two control plots without poultry litter application. Surface runoff was collected using a flume installed in a trench. Escherichia coli was quantified from sampled runoff and used as an indicator of fecal contamination by the most probable number (MPN) technique. No significant differences were observed in average event mean concentrations (EMCs) relative to storm intensity. Statistically significant differences were observed in average EMCs relative to time lag between litter application and rainfall. A nonlinear relationship was observed between average Escherichia coli EMC and time lag, with the EMC decreasing between 0 hr (1.6×105 MPN/100 mL) and 24 hr (1.3×104 MPN/100 mL) and then increasing at 120 hr (4.3×104 MPN/100 mL). Escherichia coli were always detected in the control plots (average EMC of 6.8×103 MPN/100 mL) indicating the presence and transport of fecal bacteria from sources independent of the immediate poultry litter application. Even though poultry litter application may increase E. coli concentrations in runoff, other sources of fecal contamination serve as a significant component of the total E. coli EMC, especially as the time lag between litter application and rainfall events increase.