The aggregative pattern of adherence (AA) exhibited by enteroaggregative Escherichia coli upon HEp-2 cells is a plasmid-associated property which correlates with aggregative adherence fimbria I (AAF/I) expression and human erythrocyte hemagglutination. By using cloning and mutagenesis strategies, two noncontiguous plasmid segments (designated regions 1 and 2) required for AA expression have previously been identified in enteroaggregative E. coli 17-2. TnphoA mutagenesis was performed on clones containing region 1, and 16 TnphoA mutants which were negative for the AA phenotype were analyzed. The TnphoA insertion site for each mutant was determined by junctional DNA sequencing. All 16 mutations occurred within a 4.6-kb span in region 1. Nucleotide sequence analysis of the region revealed four contiguous open reading frames, designated aggDCBA, in the same span. AA-negative TnphoA insertions into all open reading frames except aggB were obtained. On the basis of mutational analysis and protein homology data, it is inferred that aggA, aggC, and aggD are involved in biogenesis of AAF/I, encoding a major fimbrial subunit, outer membrane usher, and periplasmic fimbrial chaperone, respectively. By immunogold electron microscopy, polyclonal antiserum raised against the aggA gene product decorated AAF/I fimbriae, affirming that AggA encodes an AAF/I subunit.