Herpes simplex virus (HSV) latency can be established in vitro following arrest of virus replication and survival of infected cells in culture. Human cytomegalovirus (HCMV) has been shown to interact with HSV, resulting in reactivation of latent HSV. In addition, impaired replication of superinfecting HSV occurs in HCMV-infected human cells. HCMV-infected human embryonic lung cells inhibit production of infectious HSV despite replication of HSV DNA at levels comparable to those in control cultures infected only with HSV. Using radioimmunoprecipitation techniques, we found that the synthesis of HSV type 1 proteins of the alpha, beta/gamma, and gamma kinetic classes was impaired during the restricted replication of HSV in HCMV-infected HEL cells. However, synthesis of the HSV beta protein ICP-8 and HCMV alpha and beta proteins was not significantly affected in superinfected cell cultures.