Enterotoxigenic Escherichia coli isolated from diarrhea stools of chickens were examined for production of heat‐stable enterotoxin II which is considered to be implicated only in diarrhea of pigs. Seven out of 38 strains examined were found to contain heat‐stable enterotoxin II gene, determined by colony hybridization and the polymerase chain reaction. The culture supernatants of these strains caused fluid accumulation in the mouse intestinal loop test. This fluid accumulation activity was not lost by heating at 100°C and was neutralized by anti‐heat‐stable enterotoxin II antiserum. Purified heat‐stable enterotoxin II caused fluid accumulation in the chicken intestinal loop assay. These results indicate that STII‐producing E. coli is implicated in chicken diarrhea.