Previous reports have suggested that transient inhibition of DNA synthesis by chemicals or ultraviolet light causes some of the DNA to replicate more than once in one cell cycle, i.e., that it induces overreplication of DNA. The data that led to this suggestion were obtained from cesium chloride equilibrium density gradient analyses, in which cells were incubated with bromodeoxyuridine so that the DNA synthesized after incubation with the inhibitor could be densitometrically separated from the DNA that had been radioactively labeled before incubation with the inhibitor. An unresolved problem with these analyses was that the data also suggested that overreplication must have occurred in control cells, i.e., those not incubated with an inhibitor of DNA synthesis. We show here that the latter result is probably due to an artifact of cesium chloride equilibrium density gradient analysis, probably because of nonspecific trapping of DNA in regions of the gradients where there are large amounts of DNA. We also used another protocol that avoids this artifact; with this protocol any overreplicated DNA would be found where heavy-heavy DNA bands and nonspecific trapping cannot occur. When this protocol was used there was no evidence that transient inhibition of DNA synthesis induces overreplication of DNA.