Retrovirus-derived vectors are overwhelmingly preferred over other methods for ex vivo gene therapy because they provide permanent integration of foreign genes into cellular DNA. In comparison, cationic lipids mediate efficent gene transfer, but expression is transient. When we combined cationic lipids with retrovirus particles we obtained a significant enhancement of transduction efficiency, depending upon the type of lipid formulation and the dose used. The relative effectiveness of these cytofectins was: DOSPA:DOPE > DOTMA:DOPE > DOTAP, resulting in 60-, 37-, and 5-fold increases in transduction efficiency, respectively, at optimum dosage. The effect of polycationic DOSPA:DOPE was dependent upon the viral envelope glycoprotein, was attainable by lipid treatment of either cells or virus particles, was not enhanced by the addition of polybrene, and was inhibited by chloroquine. These results strongly suggested that DOSPA:DOPE act primarily by modulation of charge associated with the viral envelope and cell membrane, enhancing retroviral transduction, rather than by providing an alternative pathway of transfection. DOSPA:DOPE is useful for improving the efficiency of gene transfer as well as the sensitivity with which retroviruses can be detected in biological fluids.