Human intestinal lamina propria mononuclear cells have been shown to mediate mitogen-induced cellular cytotoxicity, antibody-dependent cellular cytotoxicity, and lymphokine activated killer cell function. However, although natural killer cells have been demonstrated in the gut mucosa of rodents, recent reports found little or no spontaneous cytotoxic activity in the lamina propria of the human gut. Using the natural killer cell-related monoclonal antibody NKH-1, which has not previously been applied to studies of mucosal killer cell function, we have shown by immunofluorescence that 2%-3% of enzymatically dispersed lamina propria lymphocytes are NKH-1+. A "panning" technique was then used to enrich for the NKH-1+ cells. Panned cells were consistently greater than or equal to 80% NKH-1+ by indirect immunofluorescence. Unlike their counterparts in the peripheral blood, the mucosal NKH-1+ cells were Leu-11-. Although unseparated lamina propria lymphocytes failed to exhibit natural killer activity against K562 targets in 4-h chromium release assays at effector to target ratios of up to 100:1, the NKH-1+ cells were cytolytically active at ratios of less than 5:1. Mucosal lymphocytes depleted of natural killer cells (NKH-1-) exhibited cytotoxic activity when cultured for 72 h with interleukin-2. The precursors of the lymphokine culture activated phenomenon were NKH-1-, Leu-11-, T4-, T3-, T11+, and T8+. Although lamina propria T3+ cells did not exhibit spontaneous or culture activated cytotoxicity, they were shown to exhibit nonspecific anti-CD3 (anti-T3)-induced T-cell cytotoxicity. In conclusion, functional natural killer and lymphokine activated killer cells are both present in the human gut mucosa and represent distinct populations of cytotoxic cells. In addition, anti-CD3-induced cytotoxicity is a feature of mucosal T cells. These mucosal killer cell subsets differ phenotypically from those previously described in the peripheral blood.