Multi-elemental, isotope selective nano-scale secondary ion mass spectrometry (NanoSIMS) combined with confocal laser-scanning microscopy was used to characterize the subcellular distribution of N-15-labeled cisplatin in human colon cancer cells. These analyses indicated predominant cisplatin colocalisation with sulfur-rich structures in both the nucleus and cytoplasm. Furthermore, colocalisation of platinum with phosphorus-rich chromatin regions was observed, which is consistent with its binding affinity to DNA as the generally accepted crucial target of the drug. Application of N-15-labeled cisplatin and subsequent measurement of the nitrogen isotopic composition and determination of the relative intensities of platinum and nitrogen associated secondary ion signals in different cellular compartments with NanoSIMS suggested partial dissociation of Pt-N bonds during the accumulation process, in particular within nucleoli at elevated cisplatin concentrations. This finding raises the question as to whether the observed intracellular dissociation of the drug has implications for the mechanism of action of cisplatin. Within the cytoplasm, platinum mainly accumulated in acidic organelles, as demonstrated by a direct combination of specific fluorescent staining, confocal laser scanning microscopy and NanoSIMS. Different processing of platinum drugs in acidic organelles might be relevant for their detoxification, as well as for their mode of action.