Pyronin Y forms fluorescent complexes with double-stranded nucleic acids, especially RNA, enabling semi-quantitative analysis of cellular RNA in flow cytometry, to estimate the RNA content per cell in formalin fixed EL4 leukosis tumor cells, enzyme dispersed R3327-G rat prostatic adenocarcinoma cells, mouse spleen cells stimulated with concanavalin A, and human peripheral blood lymphocytes stimulated with phytohemagglutinin. A fluorescent staining procedure based on pyronin Y is described. The technique has been used to stain RNA in human reticulocytes for subsequent flow analysis and sorting. In viable cells this dye also accumulates in mitochondria. At a concentration of 1.7 to 3.3 μM, pyronin Y is localized almost exclusively in mitochondria of cultured cells, similar to another mitochondria! probe, rhodamine 123. At that concentration PY is not toxic but suppressed cell growth, arresting cells. Pyronin Y has long been used, in combination with other dyes such as Methyl Green, as a differential stain for nucleic acids in paraffin tissue sections. In sections stained with Methyl Green-pyronin Y, red blood cells, elastic fibre of blood vessels, zymogen granules of pancreatic acinar cells, surface membrane of heptocytes and kidney tubular cells showed strikingly strong green and/or red fluorescence, while the nuclei of cells appeared non-fluorescent. Pyronin Y binds to double stranded RNA (dsRNA) including messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA).