We describe here the use of fluorescence in situ hybridization (FISH) to measure the transfection efficiency of the transient expression vector pCMVcat in lymphoblasts and fibroblasts. By using a pCMVcat probe, we can visualize the location of the plasmid after transfection and thus determine transfection efficiency. In this report, we show that, for transfection of pCMVcat by the diethylaminoethyl-dextran method, the transfection efficiency was about 15 and 70 times greater in fibroblasts and lymphoblasts, respectively, when measured by the FISH method as compared to the efficiency measured by cotransfection with pCMV beta gal. Based on these results, we conclude that the FISH method is a highly sensitive, specific and direct measure of transfection efficiency of a transient expression vector and that it may be useful for evaluating laboratory assays in which the quantitative aspects of transfection and the effect of plasmid DNA damage on transfection efficiency are important.