A HeLa cell transient-expression assay system was used to determine if isolated immediate early (alpha) genes from herpes simplex virus (HSV) could transcriptionally activate (transactivate) the type 1 (HSV-1) thymidine kinase (TK) gene [an early (beta) gene]. Cells transfected with the TK gene alone transcribed very low levels of TK RNA. Cells cotransfected with plasmids bearing the sequences that encode the alpha-gene product infected cell protein 0 or 4 (ICP0 or ICP4) and the TK gene faithfully transcribed high levels of TK RNA. The plasmid containing the sequences encoding ICP0 was a more potent transactivator than the plasmid containing the sequences for ICP4.