The helical repeat of DNA in solution has been measured directly by analyzing the gel electrophoretic patterns of pairs of covalently closed DNAs with length differences between 1 and 58 base pairs, out of a total length of about 4350 base pairs per DNA molecule. The method is based on the observation that for a covalently closed DNA of a fixed size of n base pairs (n of the order of several thousand), under appropriate conditions, two topological isomers (topoisomers) differing by 1 in their linking numbers are well resolved by gel electrophoresis. If the size of the DNA is increased to n + x base pairs, unless x is an integral multiple of the helical repeat h, the bands of the topoisomers with n + x base pairs per molecule are all shifted relative to the bands of the topoisomers with n base pairs per molecule. The magnitude of the shift is directly related to the nonintegral residual of x/n. Analysis of the set with x ranging from 1 to 58 gives the DNA helix repeat in solution as 10.4 base pairs per turn under physiological conditions, with an estimated probable error of +/- 0.1. This result strongly supports the double helix structure of DNA and rejects the side-by-side model of Rodley et al. [Rodley, G.A., Scobie, R.S., Bates, R.H. T & Lewitt, R.M. (1976) Proc. Natl. Acad. Sci. USA 73, 2959-2963]. The helical repeat of DNA measured in solution is significantly different from the value 10.0 base pairs per turn for the B form fiber structure.