Tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) exert a wide array of immunoregulatory, partly related effects. We examined the production of these two mediators by the human hairy cell leukemia cell line Eskol. Combined cell lysate and supernatant of Eskol cells (0.5 x 10(6) cells ml(-1)) incubated for 18 h, contained a mean of 1.5 ng ml(-1) TNF-alpha. This spontaneous TNF-alpha synthesis was enhanced by phorbol ester (PMA) and phytohemagglutinin (PHA) and decreased by dexamethasone. Nitrite, the stable product of NO, accumulated in the supernatant of Eskol cells after prolonged incubation. Maximal nitrite concentrations (range: 0.8-3.5 microM at 2 x 10(6) cells ml(-1)) were detected after 7 days of incubation. NO production was augmented by PHA and reduced by PMA. The inhibitors of NO synthase N(G)-monomethyl-L-arginine (L-NMMA) and aminoguanidine decreased NO synthesis. Simultaneous activation with the proinflammatory cytokines, interferon-gamma, interleukin-1beta and TNF-alpha, increased NO synthesis. These results suggest that NO production in Eskol cells results from inducible NO synthase activity. This is the first direct demonstration of NO formation in human lymphoid cells. The cell line, Eskol, may serve as a model to study regulation of TNF-alpha and NO synthesis in human B-cell leukemia.