Twenty-three healthy human adults with naturally occurring (spontaneous) mononuclear cell cytotoxicity (MCC) to cultured human tumor cells were evaluated in a serial study over 23 months to determine whether their initial MCC levels remained stable. A 3-hour 51Cr-release microcytotoxicity assay, in which the HBT-3 cancer-derived cell line with HeLa markers and the ALAB breast cancer-derived cell line were used, was standardized and its suitability for such serial studies was evaluated. The data showed that the 51Cr-release assay is reproducible and suitable for use in serial studies of MCC. The intrinsic technical error for a single assay averaged 12.5% (2-26%) coefficient of variation. The greatest sources of technical variation with time were the passage level of the target cells and the temperature at which the effector cells were stored and handled. When these technical factors were controlled, naturally occurring MCC was fairly reproducible with time. Of the healthy individuals with initial high or low MCC levels, 61% tended to retain these levels in a stable fashion, but the remaining 39% studied showed fluctuations of MCC greater than 33% coefficient of variation.