The ligated intestinal segment test in the young calf was utilized to establish the enterotoxigenicity of approximately 600 Escherichia coli isolates obtained from fecal specimens intestinal contents of calves with diarrheal disease. One hundred and fifty isolates were routinely tested in a single calf. There was little problem with false-positive reactions. False-negative reactions normally occurred only if the isolate was tested in the posterior 3 m of the small intestine. The ligated small intestine of the calf was distended by E. coli isolates of bovine, porcine, and human origin. Use of the calf ligated intestinal segment test is recommended to determine the enterotoxigenicity of E. coli isolates of bovine origin.