The herpes simplex virus type 1 thymidine kinase (tk) gene (UL23) lies upstream of the gH (UL22) gene with its 3' end overlapping the gH promoter, and it overlaps the UL24 gene's regulatory and coding sequences at its 5' end in a head-to-head orientation. Thus, tk expression could affect gH expression by promoter occlusion and UL24 expression by transcriptional or posttranscriptional mechanisms. To investigate these possibilities, we analyzed the effects of tk promoter mutations that reduce tk expression on gH and UL24 expression. For gH, tk promoter mutations did not increase the accumulation of gH mRNA or the rate of gH transcription. Thus, tk expression does not appear to down-regulate gH expression. In contrast, we found that decreased tk expression correlated with increased accumulation of UL24 mRNA, particularly a 1.4-kb transcript, at early times postinfection during peak expression of tk, but not at late times when tk mRNA levels have fallen. Results from viral co-infection experiments indicated that down-regulation of UL24 mRNA accumulation requires tk expression in cis. Nuclear run-off experiments did not detect differences in UL24 transcription rates in the mutant viruses. Although we cannot completely exclude a transcriptional mechanism for this down-regulation, these results can be explained by an antisense RNA mechanism acting preferentially in cis.