Production of coli surface-associated antigen 1 (CS1) by Escherichia coli strain E24377 of serotype O139.H28 was controlled by a plasmid that also encoded heat stable and heat labile enterotoxins and CS3. The presence of a regulatory sequence was detected on this plasmid by hybridization with the cfaD gene that regulates expression of colonization factor antigen I fimbriae and is at least 96% homologous with the rns sequence controlling production of CS1 or CS2 fimbriae by strains of serotype O6.H16 of appropriate biotype. A separate plasmid, pDEP20, carrying the structural genes for CS1 synthesis was identified and transformed into E. coli strain HB101 or a derivative of strain E24377 without large plasmids. Transformants carrying pDEP20 did not produce CS1 fimbrial antigen, but antigen expression was obtained when a cloned cfaD gene or a wild-type plasmid carrying the rns sequence was introduced. Transposon mutagenesis with Tn1000 identified a 3.7 kbp region of pDEP20 essential for production of CS1 fimbriae. Genes encoding production of CS1 fimbriae were cloned on a 9.9 kbp BamHI fragment and were expressed in the presence of the cfaD sequence. A strain producing both CS1 and CS2 antigens was constructed by introduction of the cloned cfaD gene into a strain of serotype O6.H16 biotype C carrying plasmid pDEP20.