The genes determining the biosynthesis of a new putative colonization factor, designated PCF09 have been cloned from an LT+ enterotoxigenic Escherichia coli 09:H- isolated during an outbreak of infant diarrhea in Central Australia. Electron microscopy has shown it to be of the fibrillar type. Purification of the major pilin subunit showed it to have a size of approximately 27 kDa. NH2-terminal analysis of the major subunit has shown the PCFO9 determinant to be distinct from other fimbriae although there is some conservation of certain residues. A synthetic oligodeoxynucleotide probe based on the NH2-terminal amino acid sequence of the purified protein has been used in Southern hybridization analyses to define the region on pPM1320 encoding the structural gene for the major pilin subunit.