agn43 encodes a major phase-variable outer-membrane protein, antigen 43 (Ag43), involved in autoaggregation of Escherichia coli cells. The gene is present in single copy on the chromosome of E. coli K-12. In contrast, Southern hybridization and gene inactivation studies demonstrate that control producer strain E. coli ML308-225 possesses duplicate copies of agn43 (agn43A and agn43B). Construction and analyses of single and double knockout mutants clearly show that both alleles are capable of expressing antigen in a phase-variable manner, with observed differences in the ON<-->OFF switch frequencies appearing to favour expression of Ag43B under conditions of normal laboratory growth. Comparative analysis of agn43A and agn43B gene sequences revealed 98% identity at the nucleotide and predicted protein levels, with differences in the protein sequence of the surface-expressed alpha(43) subunit altering the surface probability of one of the predicted epitopes. Analysis of a panel of enteropathogenic E. coli strains by Southern hybridization using agn43-specific gene probes provided strong evidence for the presence of varying numbers of agn43 alleles within clinical isolates. Taken together, the results indicate the presence of a family of distinct Ag43 proteins encoded by multiple chromosomal alleles.