Fecal prevalence of Escherichia coli O157 in ruminants is highest in the summer decreasing to very low levels in the winter. We hypothesize that this seasonal variation is a result of physiological responses within the host animal to changing day-length. To determine the effects of melatonin (MEL) on fecal shedding of E. coli O157:H7 in cattle, eight crossbred beef steers identified as shedding E. coli O157:H7, were allotted to treatment: control or MEL (0.5 mg/kg body weight (BW); 1x) administered orally daily for 7 days. After a 5-day period of no treatment, a second MEL dose (5.0 mg/kg BW; 10x) was administered daily for 4 days. Fecal samples were collected daily for qualification of E. coli O157:H7. No differences (P > 0.10) were observed in the percentage of E. coli O157:H7 positive fecal samples in steers receiving the 1x MEL dose, however the 10x dose decreased (P = 0.05) the percentage of fecal samples E. coli O157:H7 positive. Serum MEL concentrations were higher in the 1x, but not 10x, treated animals compared to control animals. Although it is difficult to explain, this may be a result of decreasing day-length increasing serum melatonin concentrations that may have masked any treatment effect on serum melatonin. In a second similar experiment, a second group of cattle (heifers and steers) were administered tryptophan (TRP) over a 17-day experimental period (5 g/head/day for 10 days followed by 10 g/head/day for 7 days). Tryptophan had no effect (P > 0.20) on the percentage of fecal samples positive for E. coli O157. Serum TRP (P < 0.05), but not MEL (P > 0.20), concentrations were elevated in TRP-treated animals. The decrease in the number of positive fecal samples observed in the first experiment, may be related to gastrointestinal MEL, affected by the 10x, but not 1x MEL dose.