Herpes simplex virus 1 (HSV-1) ICP27 and ICP8 proteins have both been implicated in the transcription of late genes and regulation of viral gene expression. We showed previously that ICP27 and ICP8 associate with the RNAP II holoenzyme (Zhou and Knipe, J. Virol. 76, 5893-5904). Here, we demonstrate that ICP27 and ICP8 coprecipitate from lysates of HSV-1-infected HEp2 cells and from lysates of insect cells expressing ICP27 and ICP8, the latter being in the absence of other HSV-1 proteins. By expressing and purifying hexahistidine-tagged ICP8 (His-ICP8) and maltose binding protein (MBP)-tagged ICP27 (MBP-27) proteins and performing in vitro immunoprecipitation and pull-down assays, we also demonstrate that ICP27 and ICP8 coprecipitate in the absence of other viral or cellular proteins. Taken together, these data provide evidence that ICP27 and ICP8 interact directly in vitro and in infected cells. We hypothesize that the ICP27-ICP8 interaction plays a role in the stimulation of late gene transcription.