The majority ofEscherichia colistrains isolated from urinary tract infections have the potential to express multiple fimbriae. Two of the most common fimbrial adhesins are type 1 fimbriae and pyelonephritis-associated pili (Pap). Previous research has shown that induced, plasmid-based expression of a Pap regulator,papB, and its close homologues can prevent inversion of thefimswitch controlling the expression of type 1 fimbriae. The aim of the present study was to determine if this cross-regulation occurs when PapB is expressed from its native promoter in the chromosome ofE. coliK-12 and clinical isolates. The regulation was examined in three ways: (1) mutated alleles of thepapregulatory region, includingpapBandpapI, that maintain thepappromoter in either the off or the on phase were exchanged into the chromosome of bothE. coliK-12 and the clinical isolateE. coliCFT073, and the effect on type 1 fimbrial expression was measured; (2) type 1 fimbrial expression was determined using a novelfimS : : gfp+reporter system in mutants of the clinical isolateE. coli536 in which combinations of complete fimbrial clusters had been deleted; (3) type 1 fimbrial expression was determined in a range of clinical isolates and compared with both the number of P clusters and their expression. All three approaches demonstrated that P expression represses type 1 fimbrial expression. Using a number of novel genetic approaches, this work extends the initial finding that PapB inhibits FimB recombination to the impact of this regulation in clinical isolates.