Five strains of hemolytic Escherichia coli isolated from dogs suffering from diarrhea were shown by radioactive and enzyme-labeled oligonucleotide probes to possess genes coding for heat-stable enterotoxin (STIa). Four of the strains were shown by immunoassay (enzyme-linked immunosorbent assay) and bioassay (infant mouse test) to produce STI in vitro. All five strains, however, were able to induce fluid accumulation in ligated dog intestinal loops. The four STI-producing strains all possessed the K99 fimbrial antigen (F5) and belonged to serotype O42:H37. In these strains, genes encoding STI were located on a 98-megadalton plasmid. In the fifth strain, which produced STI in vitro only after several subcultivations, the STI gene was located on an 80-megadalton plasmid. This strain was nontypable.