We have devised a method for detecting and estimating the sizes of large bacterial plasmids in the presence of genomic DNA by pulsed-field gel electrophoresis (PFGE). Bacteria harboring plasmids were embedded in agarose and lysed using a rapid protocol. Plugs were incubated with (S 1) nuclease and subjected to PFGE in agarose gels. S1 nuclease converted supercoiled plasmids into full-length linear molecules. Large plasmids migrated as discrete bands that were readily observed after ethidium staining. Their sizes were reliably estimated by comparison with linear DNA markers. Without (S 1) digestion, supercoiled plasmids migrated at rates that were not a simple function of their molecular weights, making size determinations problematic. S1PFGE detected megaplasmids up to 609 kilobases ( (\mathbf{b b}) ) in six genera of bacteria (Agrobacterium, Escherichia, Klebsiella, Pseudomonas, Salmonella, and Staphylococcus). The procedure gave size values consistent with previous estimates for characterized megaplasmids. Eight new plasmids between 102 and (316 \mathrm{~kb}) were discovered in Klebsiella and Staphylococcus. S1-PFGE avoids the difficulties of plasmid isolation, eliminates the preparation of probes, and does not require knowledge of restriction enzyme cleavage sites. It detects multiple large plasmids up to the limits of PFGE and can be used to screen for megaplasmids in many strains simultaneously. co 1995 Academic Press, Inc.