We examined the ability of a colonization factor antigen I (CFA/I) polynucleotide probe to identify coli-surface antigen 4 producing (CS4+) strains of enterotoxigenic Escherichia coli (ETEC). At low stringency (LS) the probe hybridized to colony lysates of strains previously shown to produce CS4 or CFA/I fimbriae. Only DNA from CFA/I+ strains maintained a stable probe-target hybrid under high stringency (HS) conditions. On examination of several clones from three previous CS4 producers, identified as positive in LS and negative in HS colony hybridization, spontaneous loss of nucleotide sequences homologous to a gene encoding a positive CFA/I regulator, CfaD, was found to be associated with lacking expression of CS4. Our findings indicate that, on stored or subcultured isolates of ETEC, identification of CS4 strains may benefit from applying gene probe technology.