Nine weaned calves aged from 8 to 12 weeks were fitted with rumen cannulas and were inoculated by cannula with 10(10) CFU of a five-strain mixture of nalidixic acid-resistant Escherichia coli O157:H7. Six calves were fasted for 48 h on days 15 and 16 and days 22 and 23 after inoculation. Samples of rumen contents and feces were obtained daily to enumerate E. coli O157:H7 populations and to determine rumen volatile fatty acid (VFA) concentrations and rumen pH. Fasting resulted in a marked decrease in rumen VFA concentrations from a mean of 135 mmol/liter before the fast to a mean of 35 mmol/liter during the second day of the fast. However, there was no correlation between daily VFA concentration and daily rumen or fecal numbers of E. coli O157:H7 in any of the calves. Fasting generally had no significant effect on the rumen or fecal numbers of E. coli O157:H7. The exception was a single fasted calf that experienced a 3-log(10) CFU/g increase in fecal shedding during and after the first fast. Despite the consistent changes in VFA concentrations in fasted calves, the fluctuations in rumen numbers of E. coli O157:H7 in the rumen of fasted calves were minimal. At the end of the experiment, E. coli O157:H7 was detected in either the rumen or omasum in two of three control calves at necropsy and in either the rumen or reticulum in five of six fasted calves. E. coli O157:H7 was detected in the colon in two of three control calves and in six of six fasted calves at necropsy. These results suggest that in cattle already shedding E. coli O157:H7, feed withdrawal and the associated changes in rumen pH and VFA concentrations have little effect on fecal shedding and rumen proliferation of E. coli O157:H7.