The application of poultry litter to agricultural fields can provide plant nutrients for crops and forage production, but fecal bacteria and the sex hormones estradiol and testosterone are components of litter that can be detrimental to the environment. Our objective was to determine if applications of poultry litter to small watersheds would contribute to the load of fecal bacteria and sex hormones to soil and runoff. We, therefore, investigated the fate and transport of fecal bacteria, estradiol and testosterone from surface applied poultry litter to four small watersheds. Poultry litter was applied to meet the nitrogen requirements of pearl millet (Pennisetum glaucum L.) in 2000 and grain sorghum [Sorgham bicolor (L.) Moench] in 2001. Neither Salmonella nor Campylobacter were detected in the litter but the fecal indicator bacteria were. The average load of total coliforms,Escherichia coli, and fecal enterococci applied with the litter was 12.2, 11.9, and 12.7 log10 cells ha(−1), respectively. The average load of estradiol and testosterone was 3.1 and 0.09 mg ha(-1), respectively.Runoff events first occurred seven months after the first litter application in 2000, and three weeks after the second application in 2001.Only for the 25 July 2001 runoff event three weeks after the second litter application, were the concentrations of total coliforms, E. coli,and fecal enterococci in runoff greater than background concentrations which were on average 5.2, 1.1, and 2.9 log10 MPN 100 ml(−1),respectively [corrected]. Average background levels of total coliforms, fecal enterococci,and E. coli in surface soil were 8.2, 7.9, and 3.5 log (10) cells kg(−1) soil. At the rate of litter application the concentrations of estradiol and testosterone in the litter did not appear to impact the background levels in the soil and runoff. Because concentrations of sex hormones in litter from other broiler operations are known to be greater than in the litter we applied, further study on the connection between concentrations of sex hormones in poultry litter and operational practices is recommended.