We examined the inhibitory effect of chlorpromazine, a phenothiazine derivative, on SV40 DNA replication in a HeLa cell extract. The inhibition by chlorpromazine was reversed by cardiolipin, phosphatidylinositol, phosphatidylglycerol, and phosphatidylserine. Studies on a staged reaction showed that the pre-elongation step was sensitive to chlorpromazine: chlorpromazine-resistant DNA synthesis was observed when the drug was added after the pre-elongation step. Analysis of replication products by agarose gel electrophoresis revealed that some steps of DNA chain elongation and maturation of closed circular forms were also sensitive to this drug.