The effect of various metabolic inhibitors on the expression of cytolytic activity of CD4 (TH1) and CD8 (CTL) clones was studied. The cytolytic activity of CD4 clones, but not CD8 clones, was sensitive to the RNA synthesis inhibitor actinomycin D and the protein synthesis inhibitor cycloheximide. Conversely, cholera toxin (CT) inhibited cytolytic activity of CD8, but not CD4 clones. Both mitomycin C, a DNA synthesis inhibitor, and cyclosporin A (CsA) failed to inhibit the cytolytic activity of either CD4 or CD8 clones. Although pretreatment with CsA or CT did not inhibit the cytolytic activity of CD4 clones, lymphokine (interleukin 2, IL2, interferon-gamma, IFN-gamma, and tumor necrosis factor, TNF) production was strongly inhibited. Similarly, pretreatment of a CD8 clone with actinomycin D or CsA inhibited lymphokine production without affecting cytolytic activity. The production of mRNA for TNF and IFN-gamma by concanavalin A-activated CD4 clones was also inhibited by CsA and CT. Moreover, perforin-specific mRNA was not detected in activated CD4 clones. Collectively, these observations demonstrated that de novo synthesis of RNA and protein is required for expression of cytolytic activity of CD4 clones, yet production of TNF, INF-gamma, IL 2 and perforin is not involved. In contrast, the cytolytic machinery of CD8 clones is present prior to activation and is quickly expressed following activation even when de novo synthesis of RNA, protein and lymphokines is blocked.