An alkaline phosphatase-labeled 30-mer oligonucleotide probe was designed to detect the gene for pilus colonization factor antigen III (CFA/III) of the human type of enterotoxigenic Escherichia coli (ETEC). The CFA/III probe was used to identify CFA/III-producing ETEC among 303 Escherichia coli obtained from subjects with traveler's diarrhea. Six isolates positive for the CFA/III gene were found. This result was confirmed immunologically by using a specific monoclonal antibody developed against CFA/III. These six isolates, isolated from travelers returning from India, Pakistan and China, were all positive for the gene of heat-labile enterotoxin and possessed an identical serotype (025:H-).