An investigation has been made on the properties of dimethylaminostyrylmethylpyridiniumiodine (DASPMI), its reaction with isolated pigeon heart mitochondria and its suitability as a vital strain for mitochondria in situ. DASPMI is a low toxicity specific vital stain for mitochondria in living cells. In vitro dye concentrations over 6 nmol/mg protein inhibit fast (state 3) respiration after a preincubation time of more than 5 min in the presence of substrate. No uncoupling was observed. Energization of pigeon heart mitochondria by addition of ATP or various substrated yields an average 8.5-fold increase in fluorescence intensity in relation to DASPMI-stained mitochondria that are under anoxia, substrate deficiency, or under the influence of respiratory inhibitors, or uncouplers. The alterations in fluorescence intensity are not primarily due to ion movements or pH changes. The amount of dye (2.96+/-0.8 nmol) yielding maximal fluorescence response with 1 mg mitochondrial protein remains constant during energization of mitochondria. As indicated by electron microscopic studies the observed changes in emission intensity may be related to changes in the fine structural organisation of cristae. A remarkable difference exists between isolated mitochondria and mitochondria in situ with respect to the reaction to cyanide. According to the reported results DASPMI will be a useful probe for the investigation of mitochondrial activities in living cells.