We have developed a digital image processing technique based on highpass filtering of microfluorimetric images for selective transmission of fine image details corresponding to mitochondria. This technique enabled the detection of the mitochondrial calcium signals with high selectivity, simultaneously with the cytosolic calcium signal. The validity of this technique was supported in primary cultures of rat brain capillary endothelial cells loaded with X-rhod-1 by the results that (i) inhibition of the mitochondrial Ca2+ uptake by discharging the mitochondrial membrane potential selectively abolished the transient of the highpass filtered signal evoked by ATP, and (ii) CGP-37157, a selective blocker of the mitochondrial Na+/Ca2+ exchanger, increased the peak amplitude of highpass filtered (mitochondrial) Ca2+ transients and caused a sustained plateau. The highpass filtering technique enabled the analysis of the mitochondrial Ca2+ transients in high temporal resolution. We found a uniform and monophasic rise of [Ca2+] in the mitochondrial population of the cell, following the cytosolic [Ca2+] with a delay at onset and peak. The introduced highpass filtering technique is a powerful tool in the high spatial and temporal resolution analysis of mitochondrial calcium transients, and it could be especially important in specimens where genetically targeted probes fail. Copyright 2001 Harcourt Publishers Ltd.