With an in vitro culture technique combined with light microscopy, immunocytochemistry and molecular probing, we previously detected occult tumor cells in histologically-normal human bone marrow harvested for autologous transplantation. In this study, we mixed known numbers of malignant lymphoid (Raji and CEM) or breast cancer (MCF-7) cells with normal human bone marrow cells to determine the levels at which tumor cells can be detected before and after culture. Cytocentrifuge preparations were made before culture and after 2 or more weeks of culture and examined by light microscopy. We detected contaminating lymphoma cells at a level of more than 5% before culture, and at a level of 0.01% after culture for 2 or more weeks in 2% human lymphocyte conditioned medium. Before culture, we detected MCF-7 cells at a level of 0.001% using glucose oxidase immunocytochemical staining techniques; these cells were detected at a level of 0.00001% after culture. Since, of necessity, these calibrations rations were performed using cell lines, it is likely that these results overestimate the absolute sensitivity of these methods for detection of tumor cells in patient samples. We found the glucose oxidase immunocytochemical method more specific for detecting occult tumor cells in bone marrow than the immunoperoxidase staining method because of the absence of non-specific staining arising from endogenous peroxidase in bone marrow cells which makes the interpretation of the latter difficult. We conclude that culture techniques can increase the sensitivity of detection of occult tumor cells in human bone marrow about 100-fold.