We have used chloroquine/agarose gel electrophoresis and a blot-hybridization technique to study the modulation of superhelicity of extrachromosomal DNA in mammalian cells. The high sensitivity of the procedure has allowed us to measure the change in the specific linking difference or superhelical density (sigma) of a plasmid, psvo alpha 1p3d, after its introduction into COS-7 cells by DNA transfection. Because the molecular weight of psvo alpha 1p3d is approximately the same as that of simian virus 40 (SV40) DNA, the latter can be used as a standard for estimating the average linking difference or number of superhelical turns (tau) of psvo alpha 1p3d after separation of the different supercoiled species on chloroquine/agarose gels. It was found that transfection of monkey cells with either fully supercoiled psvo alpha 1p3d isolated from bacteria (tau = -27 +/- 1, sigma congruent to -0.051) or its relaxed form after treatment with DNA topoisomerase I yields psvo alpha 1p3d samples of the same tau and sigma values of -20 +/- 1 and -0.038, respectively. The difference between the tau values of psvo alpha 1p3d and SV40 in COS-7 cells, in which both plasmids undergo rounds of replication, corresponds to an average difference of 5 +/- 1 superhelical turns. Plasmid psvo alpha 1p3d remains at this lower level of superhelicity for at least 72 hr. The distribution in linking numbers of the topoisomers of psvo alpha 1p3d isolated from transfected COS cells is also more heterogeneous than that of SV40 DNA. These results suggest that the regulation of DNA supercoiling and chromatin assembly may be closely associated with specific DNA sequences. The approach presented here should have a wide application in the study of the regulation and functional role(s) of DNA supercoiling of plasmids in mammalian cells.