The adult liver contains lymphocytes with a unique phenotypic distribution compared to blood and other organs. We have characterized a human lymphocyte population that exhibits dual T cell and natural killer (NK) cell phenotype and function, denoted natural T (NT) cells, in nine normal adult liver specimens. Flow cytometry revealed that up to 55% (mean 27%) of hepatic (but <6% of peripheral) CD3+ lymphocytes expressed CD56, CD161 and/or one or more of the killer inhibitory receptors (KIR) p58.1, p58.2, p70 and CD94. NK function was attributed to the CD3+CD56+ cells by the demonstration that hepatic, but not peripheral, CD3+ lymphocytes could be induced to lyse NK-sensitive K562 target cells, while CD56- cells from both compartments could not. Three color flow cytometric analysis of fresh hepatic cells indicated that CD3+CD56+ NT cells can be either CD8+, CD4+ or CD4 CD8-, they express alphabeta or gammadelta T cell receptors (TCR) and CD161 and KIRs, but rarely CD16. Hepatic NT cells predominantly express the mature/activated CD45RO and CD56dim phenotypes. Analysis of mRNA production by isolated NT cells indicated a preferential usage of the invariant CD1-restricted Valpha24-JalphaQ TCR. The presence of such large numbers of chronically activated NT cells provides compelling evidence that the liver has unique immunoregulatory functions.