Single strand specific mung bean nuclease was used to probe for regions of altered secondary structure in supercoiled PM2 DNA. Supercoiled DNA is cleaved greater than or equal to 10,000 times faster than the relaxed topoisomer. Catalytic quantities of enzyme convert supercoiled DNA to both nicked-circular and unit length linear forms at pH 5 but to predominantly the nicked-circular form near neutral pH. At the elevated enzyme concentrations required to cleave relaxed DNA, unit length linear DNA and smaller fragments are produced from pH 5 to 7. One nick per supercoiled DNA molecule is introduced at pH 6.6. The nicks are repairable by DNA ligase and are not strand-specific. Snake venom phosphodiesterase selectively cleaves the strand opposite the nicks, permitting restriction endonuclease mapping. The nicks occur at three specific sites. Sites at 0.75 and 0.76 map units are cleaved with equal frequency, while a site at 0.82 is cleaved less frequently. The former sites map near one of the eight known early denaturation regions of PM2 DNA, while the latter does not. Since most early denaturation sites are not cleaved, sites other than these dA + dT-rich regions may be the preferred locations of strand unwinding and separation in supercoiled PM2 DNA.