The cytotoxic activity of NK (natural killer) cells is very important in immunological surveillance against the appearance and especially the spread of malignant disease. The aim of this study was to investigate the function of this subpopulation of cells in breast cancer patients in different clinical stages of disease prior to therapy. NK cell activity was determined in breast cancer patients and healthy controls by three different methods: standard 51-chromium-release assay and by the original colorimetric uncorrected and corrected lactate dehydrogenase (LDH) release assay. A discrepancy was shown between the assays, as the uncorrected LDH assay showed, not only, much higher values, but no stage-dependent depression in NK cell activity compared to the chromium-release assay. Further analyses of separately cultured peripheral blood lymphocytes (PBL) revealed that this difference arose from an increasing, clinical stage-dependent, spontaneous LDH release from PBL of breast cancer patients. Furthermore, a stage-dependent increase in intracellular LDH activity of PBL was found, although without difference in LDH-H and LDH-M isotype ratio, compared to controls. Increased spontaneous LDH release and intracellular LDH activity was more evident in young patients, under 40 years. Correction of the original LDH-release assay for the spontaneous LDH release activity from PBL present in the assay, gave values of NK cell activity comparable to those determined by the chromium assay and indicated that breast cancer patients have a significant depression in NK cell activity which correlates with the stage-dependent increase in spontaneous LDH release. Moreover, as both assays measure the secretory, perforin-mediated, NK cell cytotoxic pathway against tumor cells, it can be concluded that the appearance of spontaneous LDH release is an indicator of cell membrane damage which not only allows the loss of LDH, but also of the components of the secretory killing pathway, resulting in NK cell dysfunction with the progression of disease. The novel findings obtained in this work reveal the association of PBL membrane damage with clinical stage of breast cancer that can, aside from reflecting NK cell depression, underlie the defect in other PBL subsets and subsequently facilitate progression of the malignant process.