Dimers, trimers and higher multimers of two 147-base pair restriction fragments called 12 A and 12B, obtained from the Mspl digest of plasmid pBR322, migrate as sharp bands in agarose and dilute polyacrylamide gels, indicating that they are homogeneous in molecular weight. However, the electrophoretic bands corresponding to multimers of the curved fragment 12A are split into sharp sub-bands in more concentrated polyacrylamide gels. The relative intensities and spacing of the sub-bands depend on the number of monomers in the multimer, the pH of the buffer, and the presence or absence of divalent cations in the solution. Since band splitting is not observed for the normal 12B multimers under any gel-running conditions, the sub-bands observed for multimers of the curved fragment 12A must be attributed to conformational isomers which are in slow exchange on the electrophoretic time scale. Band splitting is also observed for multimers of a curved DNA fragment containing the kinetoplast bending locus and for plasmid pUC19 linearized by digestion with certain restriction enzymes. Plasmid pUC19 contains two nearly equidistant regions of intrinsic curvature (Strutz, K., Stellwagen, N. C., Electrophoresis 1996, 17, 989-995). Hence, DNA molecules containing two or more regions of curvature exist as discrete subpopulations of conformational isomers which can be observed as separate bands migrating in polyacrylamide gels.