The natural killer (NK) cell activities of spleen, thymus, bursa, peripheral blood and gut intraepithelial lymphocytes (IEL) from FP and SC chickens were investigated in 4-hr and 16-hr 51Cr release assays. Target cells were 4 different tumor cell lines derived from either an avian leukosis tumor transplant (LSCC-RP9, LSCC-RP12) or from Marek's disease lymphomas (MDCC-MSB-1, MDCC-CU36), Great variability in cytotoxic potential was observed among NK cells of different lymphoid organs. NK cell cytotoxicity varied depending upon the type of effector cells, type of target cells, the ratio of effector to target cells, and the age and genetic background of chickens. Substantial levels of NK cell activity were detected in splenn and gut IEL of SC chickens in a 4-hr assay. In contrast, the NK cytotoxicity in gut IEL of FP chickens was not detectable until 16 hr after incubation. The ranges of target cell specificity demonstrated by IEL, spleen, thymus and bursa NK cells were similar to one another and, in general, the level of cytotoxicity increased with incubation time. Thymus and bursa NK cell activity of both SC and FP chickens was not detectable in a 4-hr assay but substantial NK cell activity was demonstrated in a 16-hr assay. The results of the present study demonstrate that various lymphoid organs of chickens, such as spleen, thymus, bursa, and gut intraepithelium, contain subpopulations of cells that can mediate spontaneous cytotoxicity.