Early apoptosis is invariably accompanied by a disruption of inner mitochondrial transmembrane potential (delta psi m). Cationic lipophilic fluorochromes, such as 3,3' dihexyloxacarbocyanine iodide (DiOC6(3)), rhodamine 123, or 5,5', 6,6'-tetrachloro-1,1', 3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1), can be used to measure such an apoptotic delta psi m dissipation. However, these dyes are afflicted by the handicap that cytofluorometric analyses must be performed ad hoc on nonfixed, metabolically active cells. Here, we show that chloromethyl-X-rosamine (CMXRos) is a viable alternative to other delta psi m-sensitive probes, and that it allows for formaldehyde fixation of cells before analysis. Using this fluorochrome, we developed a three-color staining technique in which two fluorochromes (fluorescein isothiocyanate and phycoerythrin) coupled to antibodies are employed to determine expression of cell-surface antigens, and CMXRos is used to measure delta psi m. In addition, we developed an approach to assess simultaneously delta psi m and expression of intracellular antigens. Thus, the expression of Bc1-2, a mitochondrial outer-membrane protein, can be determined after staining with CMXRos, fixation, and cell permeabilization. CMXRos labeling can also be combined with determination of apoptotic DNA fragmentation using the Tunel technique. In conclusion, CMXRos provides several methodological advantages over other, nonfixable fluorochromes used for delta psi m determination.