Interferon (IFN) production by spleen cells from normal mice, mice acutely infected with Semliki Forest virus (SFV) or mice immune to SFV was measured after stimulation in vitro with either infectious or inactivated SFV. All three classes of spleen cells made IFN-alpha beta in response to infectious SFV. Spleen cells taken from mice late, but not early, after infection, or from immune mice, made IFN-gamma in response to inactivated SFV. Amounts of INF-gamma and IFN-alpha beta were similar. Normal spleen cells made no IFN (of any type) in response to inactivated SFV. The cell type producing IFN-alpha beta appeared to be the macrophage, whilst both T-lymphocytes and macrophages were necessary for IFN-gamma production. During the acute infection, the ability of spleen cells to lyse both virus-infected and uninfected target cells arose earlier than the ability to produce IFN-gamma. However, cytotoxicity towards uninfected cells fell to near background levels by day 7, whilst cytotoxicity towards infected targets remained high at that time, when IFN-gamma production was at its peak. IFN-gamma production is therefore temporally associated with cytotoxicity specifically directed against virus-infected targets, and the ability to produce IFN-gamma is a late response to SFV infection.