Previously, we demonstrated that human anti-HSV CTL and allo-antigen-specific CTL were inhibited in lysing their normally sensitive target cells when they were exposed to human fibroblasts (FB) infected with herpes simplex virus (HSV). In this study, the mechanism of inhibition of CTL lytic function by FB infected with HSV-1 (HSV-FB) was studied. CTL exposed to HSV-FB early (2 h) in the infection cycle were inhibited by a mechanism that appears to be distinct from the inhibition of lytic function mediated by HSV-FB at late times (20 h) during the infection cycle. The inhibition of CTL-mediated lysis by FB infected with HSV-1 for 2 h required the expression of ICP4, an immediate-early protein of HSV-1, but not the production of infectious virus or virus-induced shut-off of host protein synthesis. In contrast, the expression of HSV-specific glycoproteins essential for viral infectivity (glycoproteins B, D, H, K, and L), and thus, infectious virus, was required for inhibition of CTL lytic function by FB infected with HSV-1 for 20 h. Further, CTL exposed to FB infected with HSV-1 for 20 h expressed HSV-specific proteins indicating that they were infected with HSV-1. Cell-to-cell spread of HSV-1 appeared to be the major mode of transmission because 1) an insufficient level of HSV-1 was present in the supernatant of HSV-FB to inhibit CTL lytic function; and 2) paraformaldehyde-fixed HSV-FB did not inhibit CTL-mediated lysis. The inhibition of CTL lytic function by HSV-FB may be an important mechanism of HSV-induced immunosuppression, permitting the virus to spread and persist in immunocompetent hosts after primary infection or reactivation of latent HSV.