Covalently closed bacteriophage PM2 DNA samples containing varying numbers of superhelical turns were used as substrates for the holo and core enzymes of Escherichia coli RNA polymerase, E. coli DNA polymerase I, pancreatic DNAase, and two single strand-specific endonucleases from Neurospora crassa and Mung bean. The results indicate that the superhelical turns influence the properties of DNAs as enzyme substrates in two major ways: (1) if the interaction between a DNA and an enzyme involves the unwinding or winding of the DNA helix, the interaction is strongly affected by the degree of superhelicity of the DNA, since the higher free energy of a twisted DNA favors the reduction of superhelical turns. For phage PM2 DNAs with negative superhelical density, − σ 0 less than about 0.04, this factor is the dominant one. In this range of superhelical density the sensitivity of the DNAs toward endonucleolytic attack by the single strand-specific nucleates increases only slightly with increasing − σ 0. The magnitude of transcription by E. coli RNA polymerase holoenzyme, which is known to unwind the DNA helix slightly, increases rapidly with increasing − σ 0. A similar increase in template activity with increasing − σ 0 has been observed with the core enzyme, and is interpreted as evidence that the productive binding of the core polymerase also requires an unwinding of the DNA helix. (2) When − σ 0 is greater than about 0.04, it appears that the DNA is very tightly wound. Such tight twisting might reduce the binding and/or hinder the movement of certain macro-molecules along the helix. Thus, in this range of superhelix density, the template activity appears to decrease with increasing − σ 0. While the sensitivity of the DNA toward endonucleolytic scission by pancreatic DNAase is independent of σ 0, the sensitivity toward endonucleolytic scission by the single strand-specific nucleases increases markedly with increasing − σ 0 in this range. This is interpreted as resulting from large distortions of the double helix at the sharp bends at which the DNA doubles on itself because of the tight twisting. Such distorted regions become favored sites for the single strand-specific nucleases. For the DNA samples with − σ 0 as high as 0.06, there is no evidence that there is a significant fraction of bases in the single-stranded form. In the presence of oligo-nucleotides as initiators, no significant amount of DNA synthesis on twisted PM2 templates is detected with E. coli DNA polymerase I. While the highly twisted DNA with − σ 0 = 0.06 is nicked by the Mung bean enzyme about 75-fold faster than untwisted PM2 DNA, the rate is still only a few tenths of 1% of that with a single-stranded DNA as the substrate.