Liver-derived lymphocytes were isolated from 40 human livers with end-stage liver disease that were removed at the time of orthotopic liver transplantation. In addition, 10 resection specimens or whole livers removed from patients with liver cancer and seven normal livers (unused donor organs) were studied as controls. Liver-derived lymphocytes were isolated from enzymatically digested tissue by gradient centrifugation and adherence to plastic. Their phenotypical characteristics were studied by two-color flow cytometry, and effector cell function was determined in 4-hr 51Cr-release assays against a natural killer-sensitive target, K562 (natural killer activity), natural killer-resistant Daudi line (lymphokine-activated killer activity) and by P815 line with or without phytohemagglutinin to assess lectin-dependent cellular cytotoxicity. Liver-derived lymphocytes isolated from normal liver contained equal proportions of T and natural killer lymphocytes (mean natural killer/T ratio = 0.7). CD3−CD56+ CD16− natural killer cells were the main natural killer subset present in liver-derived lymphocytes, in contrast to the predominant natural killer phenotype in the circulation (CD56+ CD16+). Control liver-derived lymphocytes had levels of cytotoxicity significantly greater than those of the normal peripheral-blood lymphocytes against all three targets. In contrast, liver-derived lymphocytes isolated from organs with advanced liver disease differed markedly in the natural killer/T cell ratio and levels of liver-derived lymphocyte cytotoxicity. Liver-derived lymphocytes obtained from hepatocellular carcinoma or rejecting allografts treated by immunosuppressive therapy had virtually no cytotoxicity. The highest levels of cytotoxicity, including spontaneous lymphokine-activated killer activity (which is not normally detectable in freshly isolated peripheral-blood lymphocytes) were observed in liver-derived lymphocytes obtained from patients with non-A, non-B hepatitis and metastatic liver cancer. These liver-derived lymphocytes also had the highest natural killer/T cell ratio of all liver-derived lymphocytes in patients with liver disease studied. Alterations in natural killer numbers and function of liver-derived lymphocytes, which are seen in various liver diseases, may reflect different pathological processes, disease origins or local activation states. Functionally active natural killer cells are present in the liver even in cases of end-stage disease and may play a pathophysiological role in viral and neoplastic liver disease.