We read with great attention the letter of Diaz et al. concerning nuclear dyes and cytoplasmic staining. We are not sure if the reported observations refer to living cells or to cells undergoing apoptosis. It was previously reported that chromatin structure in living cells does not allow DNA intercalation; thus, only by increasing intracellular NaΟ© (i.e., by cell fixation or when cell death occurs spontaneously) does chromatin structure allow DNA staining of intercalating dyes (1). Ethidium bromide (EB), acridine orange, and many other nuclear dyes stain RNA (which is not protected by chromatin structure), both in the cell nucleus (particularly in nucleoli) and in the cytoplasm. Thus, in living cells, EB fluorescence is mainly dependent on RNA staining. In lymphoid cells and particularly in freshly isolated peripheral blood mononuclear cells (PBMC), cytoplasm and organelles are very scarce and nucleoli are not present. Due to this fact, EB fluorescence is not detectable in living PBMC by image cytometry. On the contrary, in cells undergoing apoptosis the chromatin structure is altered (2) and makes DNA available for intercalation (3); thus, fluorescence is induced in the cell nuclei of EB-stained PBMC undergoing apoptosis. It is common thinking that DNA staining with intercalating dyes such as EB is similar to that in the Trypan blue exclusion test. The plasma membrane in living cells is impermeable to the dye, but in dead cells it allows uptake of the dye with consequent nuclear staining. In addition to this obvious mechanism, present and past observations (1) demonstrate that EB uptake also occurs in living cells. This fact highlights the importance of changes in chromatin structure as being responsible for EB-staining of cells undergoing apoptosis, particularly in the early steps of the apoptotic process.