The effect of pulsed fields on sequencing gel electrophoresis is investigated, using DNA fragment markers ranging in size from 20 to 6557 bases. For high continuous electric fields (5000 V/55 cm) band inversion is observed in which fragments larger than 4000 bases migrate faster than those of 800-1000 bases. The use of one-dimensional pulsed field gel electrophoresis (ODPFGE) eliminates band inversion and extends the monotonic size-mobility relationship of the DNA markers up to about 4000 bases. The relevance of these results, obtained using a manual sequencing process with autoradiographic detection, to automated sequencers is discussed.