We have shown in this paper that agarose gel electrophoresis can be used to examine the tertiary structure of supercoiled SV40 DNA. Differences as small as one superhelical turn can be detected by this method. When used as an assay, it enabled us to purify from human tissue culture cells a protein which acts on supercoiled DNA, leading to a gradual removal of its superhelical turns. Using in turn the purified DNA relaxing protein to generate reaction intermediates of DNAs with different numbers of superhelical turns, we deduced that native SV40 DNA contains a minimum of 20 physical superhelical turns. The upper limit of this number is less accurately defined, but we think that it is not higher than 24. These numbers agree within very close limits with the numbers determined previously by various indirect physical methods.