TB/C3 hybridoma cells were transected with either pEF-MClneopA or pEF bcl2-MClneopA vectors to produce a control cell line (TB/CB pEF) and a cell line that overexpresses the "antiapoptotic" human bcl-2 protein (TB/C3 bc12). Flow cytometry analysis of intracellular bcl-2 protein levels enabled near on-line monitoring of the stability of bcl-2 expression in the absence of drug selection. It was possible to maintain spontaneous selection of cells with the overexpression of bcl-2 protein during semicontinuous cultures at very low dilution rates, where cells were subjected to the selective conditions of nutrient limitation and high toxic metabolite concentrations. Interestingly, cells that overexpressed bcl-2 were adapted to suspension culture conditions significantly faster than control cells. Dual fluorescence staining with acridine orange and propidium iodide allowed for discrimination between viable, apoptotic, secondary necrotic, and necrotic cells, respectively. Compared with the usual trypan blue method of establishing culture viability, dual staining demonstrated that under stressful conditions a significant proportion of cells that excluded trypan blue were also undergoing cell death through apoptosis. In batch cultures the overexpression of bcl-2 more than doubled the membrane intact (MI) cell productive period (the integral of MI cell density with respect to culture time) and increased the monoclonal antibody (mAb) production by approximately 40% when com pared with the control cell line. The overexpression of bcl-2 protein also significantly extended the cell integrity and viability by the suppression of apoptosis in conditions of hypoxia, hyperoxia, glutamine deprivation, glucose deprivation, and serum limitation. The suppression of apoptosis in anaerobic conditions suggests that bcl-2 exerts its antiapoptotic activity by a mechanism that does not involve an oxidative reactive pathway. In conditions of excess thymidine, which suppressed cell proliferation, Ml cell density and specific mAb productivity were further enhanced by the overexpression of bcl-2, which suggests the possibility of accomplishing a controlled proliferation in immortalized cell lines without invoking cell death. Cell size and intracellular mAb were increased for TB/C3 bc12 cells compared with TB/C3 pEF control cells when analyzed by flow cytometry.