Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disease associated with immune system hyperactivation and the appearance of serious systemic disturbances. The purpose of this study was an assessment of natural killer (NK) cell disturbances in a group of children with clinical signs of HLH. A total of 43 children with HLH and 17 healthy children were enrolled in the study. NK phenotyping, intracellular perforin staining, and cytotoxicity tests were performed by using the flow cytometry method. HLH patients were divided into 6 HLH types: 9% infection-related HLH; 7% malignancy-related HLH; 21% macrophage activating syndrome; 12% familial hemophagocytic lymphohistiocytosis; 2% X-linked lymphoproliferative syndrome; and 49% as HLH of unknown background. A positive correlation was observed between cytotoxicity and NK cells in children with HLH (P=0.01). In all HLH groups, the percentage of NK cells was significantly lower than in the control population. The spontaneous cytotoxicity was significantly lower in HLH patients. The results presented in this study indicate the importance of impaired function and the number of NK cells in the pathogenesis of HLH. Nonetheless, the background of disturbances seems to be different in various cases.