The herpes simplex virus type 1 (HSV-1) alpha protein ICP0 trans-activates HSV-1 early genes in transient expression assays. To investigate the function of ICP0 during HSV-1 infection, we have lowered the level of ICP0 by use of a recombinant plasmid that has been engineered to express the antisense message. Cell lines were constructed which stably carry the antisense plasmid. Total protein profiles from infected antisense cell lines showed that the level of ICP0 was reduced to less than 10% of the wild type level in two of the cell lines. However, reducing the level of ICP0 did not have a significant effect on the expression of HSV-1 early or late genes. The polypeptide patterns for the remaining infected cell polypeptides were similar in that no bands were absent although there were some quantitative differences. The level of two early proteins, glycoprotein B and glycoprotein D was reduced in one of the cell lines, however, levels were nearly equivalent to the control infection for two other cell lines tested. Virus yields were the same for the antisense cell lines and for parent cells. Decreased ICP0 levels did not lead to more restrictive phenotypes for an alpha 4 or alpha 27 mutant as protein patterns were similar for these mutants in antisense and parent cells. Therefore, while ICP0 has been demonstrated to be a strong inducer of gene expression in transient expression assays, it does not appear to have a major role as an activator during the productive infection of tissue culture cells.