It has been reported that secondary pulsed field gel (SPFG) electrophoresis can dramatically increase the speed of separation of large DNA molecules without a decrease in resolution (Zhang, T. Y., Smith, C. L., and Cantor, C. R. (1991) Nucleic Acids Res. 19, 1291-1296). However, our attempts to duplicate previous SPFG conditions were unsuccessful. We therefore sought to more precisely define the effects of secondary pulsing on the separation of large DNA and to determine the value of the technique in separating molecules up to 1100 kb. Here we report on two of the key SPFG parameters, namely the frequency and duration of the secondary pulse on the migration and resolution of DNA in SPFG. We found that the size range of separation is determined by the sum of the duration of the primary and secondary pulses. Under optimal conditions, a 25-70% increase in velocity can be achieved without loss in resolution.