The aim of the present study was to determine whether, in indo 1 acetoxymethyl ester (AM)-loaded rat cardiomyocytes, it was possible to remove cytosolic but not mitochondrial indo 1 by promoting loss of cytosolic indo 1 through plasma membrane anion pumps (which are blocked by probenecid). Isolated rat ventricular myocytes were loaded with indo 1-AM under conditions (15 min at 30 degrees C) in which about half of the dye is located within mitochondria. Cells were then maintained at 25 degrees C for 2.5 h followed by incubation at 37 degrees C for 1.5 h. After this "heat treatment," the myocyte fluorescence signal was 44% of the value of cells measured before heat treatment, and loss of fluorescence was prevented by 1 mM probenecid. The remaining fluorescence was shown to originate from mitochondria, since 1) Ca2+ uptake and efflux could be inhibited by ruthenium red and clonazepam, respectively, and 2) low concentrations of digitonin, which release only cytosolic marker enzymes, decreased fluorescence of untreated myocytes but had little effect on the fluorescence signal of heat-treated cells. We conclude that heat treatment selectively removes cytosolic indo 1, leaving a signal due to mitochondrial indo 1 only.