The influence of cations, temperature, and stem length on the supercoil-induced transition from the linear form to the cruciform state at certain inverted repeats of pVH51 and pBR322 was investigated. In general, conditions which stabilize duplex DNA over single-stranded DNA shifted the transition to higher negative superhelical density values due to an increase in the unfavorable free energy of cruciform formation. Specifically, increasing sodium or magnesium ion concentrations brought about a corresponding increase in the negative superhelical density required to cause cruciform formation at the major inverted repeat of both plasmids. A notable exception was the inverted repeat found in both of these plasmids (at position 1009 of pVH51 and 3123 of pBR322) for which Mg(II) concentrations between 1 and 5 mM brought about a lowering of the negative supercoiling required to cause cruciform extrusion at this site, suggesting a specific complex between the cruciform and magnesium. Increasing temperatures from 15 up to 45 degrees C for the pVH51 major inverted repeat and 37 degrees C for that of pBR322 shifted the transition to lower negative superhelical densities. Further increases brought about a shift to higher negative densities. For the two inverted repeats examined within pVH51, various divalent metal ions and spermidine resulted in the following hierarchy: Mn(II) less than Zn(II) less than Mg(II) less than Co(II) less than spermidine, where the transition midpoint was at lowest negative density values for Mn(II) and highest for spermidine. This hierarchy agrees qualitatively with the relative affinity of the cations for DNA-phosphates versus the bases. The influence of stem length on the supercoil-induced transition to the cruciform state was studied by in vitro deletion of portions of the pVH51 major inverted repeat. Decreasing the stem length from 13 to 10 base pairs (bp) had no effect on the ability of this sequence to adopt the cruciform state. However, a further reduction of 3 bp to give a stem length of 7 bp completely abolished the ability of this region of DNA to exist in the cruciform state, at least up to a density of -0.15. Thus, a very sharp dependency on stem length exists for cruciform formation within an inverted repeat region possessing a potential loop of five nucleotides.