The comparative efficiencies of polyornithine, CaCl2 and DEAE-dextran in enhancing the infectivity of exogenous herpes simplex virus (HSV) type 2 DNA were examined. CaCl2 was 12-times more effective in promoting genetic expression of viral DNA than DEAE-dextran, while polyornithine did not mediate any HSV DNA infectivity. A comparison of sedimentation profiles of DNA extracted from cells inoculated with viral DNA in the presence of each facilitator revealed that input 56 S HSV DNA underwent marked alteration with time. There was also extensive processing of a 20 S DNA species. The data indicated that the biological efficiency of each facilitator was related to the amount of 20 S DNA remaining at the final time periods. One possible explanation for the efficiency of CaCl2 as a facilitator was that it allowed for the reutilization of the 20 S DNA species during HSV replication. The persistence of the 20 S peak in cells treated with DEAE-dextran was a measure of the decreased efficacy of this facilitator. Finally, the absence of a 56 S peak and the greatly elevated levels of 20 S DNA at final time points in polyornithine-treated cells accounted for the failure of this compound to promote HSV DNA infectivity.