The effect of gel pore size on the anomalous mobility of certain curved DNA restriction fragments in polyacrylamide gels was studied by comparing the electrophoretic mobilities of normal and anomalous fragments in gels of varying composition but constant acrylamide concentration. Molecular weight ladders were prepared from two 147 base pair G-C rich restriction fragments, called 12A (anomalous) and 12B (normal), obtained from the MspI digestion of plasmid pBR322. The electrophoretic mobilities of multimers of the two fragments increasingly diverged with increasing molecular weight. The anomalous mobility of fragment 12A was essentially independent of gel pore size, regardless of whether the pore size was varied by increasing the acrylamide concentration at constant cross-linker concentration or by increasing the cross-linker concentration at constant acrylamide concentration. The anomalous mobility of higher multimers of fragment 12A decreased with increasing gel pore size when the pore size was varied by changing the gel concentration. However, when the gel pore size was changed by varying the cross-linker concentration at constant acrylamide concentration, the anomalous mobility of higher multimers of fragment 12A went through a maximum and then decreased as the pore size was increased. Copolymerizing acrylamide with high molecular weight linear polyacrylamides had no effect on the anomalous mobility of the 12A multimer ladder, even though the apparent absolute mobilities of all fragments increased markedly. Only by incorporating charged residues into the gel matrix or by copolymerizing acrylamide with the intercalator ethidium bromide could the difference in mobility between the 12A and 12B multimer ladders be substantially reduced or eliminated. Similar results were observed with a molecular weight ladder containing 78 base pair repeats of the bending locus of kinetoplast DNA. These results suggest that pore size alone is not responsible for the anomalously slow migration of curved DNA molecules in polyacrylamide gels.