The dynamics of single DNA molecules undergoing steady-state and pulsed-field gel electrophoresis have been studied using T2 DNA molecules labeled with acridine orange and visualized with a fluorescence microscope. It was observed that DNA molecules moving through the gel display an extension-contraction motion, and they tend to bunch up in their leading ends. Furthermore, the molecules often remain hooked around obstacles assuming U-shape conformations and then slip off, with the longer arm driving the chain. In field-inversion gel electrophoresis molecules of intermediate size show a substantially reduced mobility compared to both small- and large-size molecules. In orthogonal field alternating gel electrophoresis (OFAGE) the formation of kinks is promoted when time is allowed between the application of the two orthogonal fields so that the molecule attains a partially relaxed configuration. The formation of kinks provides a significant retardation of the reorientation of the molecules and appears to play an important role in the fractionation attained with OFAGE. © 1990 Academic Press, Inc. All rights reserved.