The detection of mitochondrial DNA (mtDNA) in living human cells could be useful for understanding mitochondrial behaviour during cellular processes and pathological mtDNA depletions. However, until now, human mtDNA has not been visualized in living cells with fluorescence microscopy, although it has been easily detected in organisms with larger mtDNA. Previous reports have stated that mtDNA staining results in homogeneous fluorescence of mitochondria or that animal mitochondria are refractory to DAPI staining. This paper shows that mtDNA of cultured green monkey kidney CV-1 can be stained using a very low concentration of DAPI, then detected by a cooled Photometrics CCD camera with 14-bit resolution detection. Indeed, under these conditions CV-1 cells have small fluorescent spots in the cytoplasm that colocalize with mitochondria, even after mitochondrial movements, uncoupling by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone and swelling. These observations have been reproduced for the human fibroblast foreskin cell line HS68. These results and known properties of DAPI as a specific DNA stain strongly suggest that mtDNA can be detected and visualized by fluorescence microscopy in human living cells, with potential developments in the study of mtDNA in normal and pathological situations.