The role of Natural Killer (NK) cells, Lymphokine Activated Killer (LAK) cells and the induction of cytokines in the hepatocellular injury of hepatitis A were studied in vitro using a 51Cr release assay in hepatitis A virus (HAV) infected MRC-5 cells (MRC-HAV). When fresh peripheral mononuclear cells (PBMC) from healthy, anti-HAV antibody (-) and (+) human donors, or patients with acute hepatitis A were used as effector cells, MRC-HAV were lysed more extensively than uninfected cells. Similarly, in models using recombinant interleukin-2 (rIL-2) pretreated PBMC from HAV antibody (-) and (+) donors or patients with hepatitis A as effector cells, MRC-HAV were lysed more extensively than uninfected MRC. Both fresh PBMC and rIL-2 pretreated PBMC from anti-HAV antibody (+) donor lysed MRC-HAV cells more effectively than PBMC collected from anti-HAV antibody (-) patients. PBMC from patients with hepatitis A during the acute phase demonstrated more severe lysis of both uninfected MRC and MRC-HAV target cells. However, MRC-HAV lysis was enhanced to a greater degree. In an attempt to identify which cells were involved in cell lysis, cytotoxicity assays were performed by separating PBMC by cell sorter using monoclonal antibodies against all T cells, NK and B-cell fractions and culturing each with supplemental rIL-2. The NK cell fraction selectively destroyed MRC-HAV, but the lytic activity of both the pan T-cell and B-cell fractions was too weak to demonstrate any significant difference. Therefore, NK-LAK cells appeared to play a more significant role in cytolysis than did T-LAK cells. Morphologic observations of cellular damage were accomplished with PBMC obtained from healthy anti-HAV antibody (+) donors. PBMC were suspended in media containing rIL-2 and incubated for 4 days on monolayers of uninfected MRC and MRC-HAV. IFN-alpha and IFN-gamma in the supernatant of the cultured media were simultaneously assayed. Severe damage to HAV-infected cells was observed. Moreover, strong induction of IFN-alpha and IFN-gamma was found with high levels measured in the supernatant. Therefore, it is likely that non-specific immune mechanisms involving NK and LAK cells play a central role in hepatocellular damage prior to the initiation of damage due to Cytotoxic T Lymphocytes (CTL).