Infectivity of herpes simplex virus (HSV) DNA was assessed by employing the calcium-phosphate transfection technique described by Chen and Okayama, originally applied to increase the efficiency of plasmid transfection by N, N-bis (2-hydroxyethyl)-2-aminoethane sulfonic acid (BES). The experimental conditions and efficiency of this transfection procedure were evaluated comparing the viral progeny titers obtained by the Chen and Okayama transfection method using DNA from wild-type strains of HSV-1 and HSV-2, as well as from mutant strains, with the viral progeny obtained by the most widely used transfection technique introduced by Graham and van der Eb. Furthermore, recombinant virus production was evaluated in marker transfer and marker rescue experiments, comparing both transfection techniques, using DNA fragments cotransfected with whole viral DNA into African green monkey (Vero) or rabbit skin (RS) cells. The viral production obtained from HSV-DNA transfected cells was enhanced approximately 1000-fold when the Chen and Okayama procedure was applied.