In acute pyelonephritis, bacterial resistance to co-trimoxazole predicts treatment failure, but the clonal basis of such resistance is undefined. We did molecular and serological analyses of 170 Escherichia coli urine isolates obtained in 1994-96 from women with acute pyelonephritis. 12 (7%) of the pyelonephritis isolates were in clonal group A (CGA; responsible for 38-51% of co-trimoxazole resistance in acute cystitis), including ten (34%) of 29 isolates that were resistant to co-trimoxazole. CGA isolates were obtained from diverse locations across the USA and were related to the O15:K52:H1 clone of the 1986-87 outbreak in London, UK. Thus, CGA is broadly disseminated and contributes to co-trimoxazole resistance in pyelonephritis as well as in cystitis.