The circular permutation assay has been used to characterize sequence-dependent bending in plasmid pUC19 (2686 bp). Linear, permuted sequence isomers of pUC19 exhibit different mobilities in large-pore polyacrylamide gels, suggesting that the plasmid contains two sequence-dependent bends, one located at similar to806 bp, close to the start site of transcription, and the other at similar to2617 bp, near the promoter of the ampicillin resistance gene. The mobility patterns are independent of the buffer in which the gels are cast and run, independent of pH over the range 6.5-8.6, and independent of the presence or absence of various monovalent ions added to the buffer, suggesting that the maxima in the mobility patterns correspond to stable bends in the helix backbone, not anisometric flexibility. Although the mobility patterns are unchanged when Ba++ or Zn++ ions are added to the buffer, several of the sequence isomers exhibit multiple sharp sub-bands in solutions containing Mg++ or Ca++ ions, indicating the presence of multiple conformational isomers. In addition, the mobility maximum near the start site of transcription is shifted by similar to100 bp in the presence of Ca++ ions, suggesting that the location of the actual bend center is somewhat variable, depending on the ionic composition of the surrounding medium. The number of maxima observed in the mobility pattern can be increased by adding a bend from another plasmid, such as Litmus 28; the number of maxima can be decreased by removing one of the bends in pUC19 by restriction enzyme digestion and religation. Hence, the number of maxima in the mobility pattern is an accurate indication of the number of stable, sequence-dependent bends present in the plasmid. The combined results indicate that the circular permutation assay in large-pore polyacrylamide gels is a reliable method of detecting and analyzing sequence-dependent bends in kilobase-sized DNA molecules.