A more convenient assay is needed for the three recognized allelic variants of papG, which encode the Gal(alpha 1-4)Gal-binding adhesin molecules of P fimbriae of uropathogenic Escherichia coli. The development and validation of a novel multiply primed polymerase chain reaction (PCR) assay is described. The assay was 100% sensitive and specific for the three papG variants, whether present individually or in any combination. PCR products specific for the "class I" (papGJ96), "class II" (papGIA2), and "class III" (prsGJ96) alleles of papG could be resolved by size in the same lane using agarose gel electrophoresis after simultaneous amplification in a single tube by multiply primed PCR. This new papG PCR assay should aid future molecular epidemiologic studies that assess the contribution of the three variants of papG to the pathogenesis of urinary tract infection.