Due to its spectral characteristics, the fluorochrome nonyl acridine orange (NAO) (lambda abs:489 nm, lambda em:525 nm), which is spontaneously incorporated by mitochondria with a high relative specificity, provides a new probe for the in situ study of these organelles by flow cytometry. In 15 min at 20 degrees C, the dye at 4.75 X 10(-6) M saturates the mitochondrial binding sites present in 1.5 X 10(6) cells. Unlike Rh 123, the fixation of the probe is not affected by the action of uncouplers and ionophores. Unlike acridine orange, its binding is not sensitive to nucleases. By studying the mitochondrial incorporation of the fluorochrome during the cell cycle of murine splenocytes, it was possible to show that the biogenesis of NAO-stained mitochondrial constituents mainly occurs during the G1 phase.