A study was conducted to determine whether intestinal fluid collected from various portions of bovine intestine differed in its effect on production of K99 pili by Escherichia coli. The small and large intestines of 7 calves, euthanatized 4 hours after a final feeding of milk, were divided into 6 to 9 segments from which intraluminal fluids were collected. Depending on the amount of fluid collected, up to 20 E coli strains that express K99 pili were grown on media prepared from the content of each specimen and then were tested for K99 pilus expression. In general, intestinal fluid from the most proximal small intestinal segments were more suppressive to K99 pilus expression than was fluid from more distal segments of small intestine. Only about 20% of the E coli test strains expressed K99 pili when grown on medium prepared from proximal small intestinal segmental fluid, whereas greater than 90% did when grown on medium prepared from distal small intestinal segmental fluid. Fluid from the large intestine varied considerably from calf to calf in its effect on K99 pilus expression. A correlation was found between K99 pilus expression and pH of the intestinal fluid, with the lower pH values (characteristic of proximal intestinal segmental fluid) being suppressive. The correlation between K99 pilus production and the pH of the medium was verified, using defined laboratory media adjusted to various pH values. Strains of E coli grown in medium at or below pH 5.5 failed to express K99 pili, whereas the same strains when grown in medium at or above pH 6.5 expressed K99 pili in abundance.