In this paper, the potential of five pyridine or phenanthrolinecontaining macrocycles as mitochondria and ER fluorescent probes is thoroughly explored. The cellular toxicity of the compounds is evaluated on breast cancer cells, non-cancerous human dermal fibroblasts and human adult dermal skin fibroblasts from a breast cancer GW-572016 231277-92-2 patient by MTT assay. The apoptosis induced on MCF-7 and NHDF cells and the MMP are evaluated by flow cytometry. The compounds ability to penetrate the intracellular medium and their localization is accomplished by fluorescence microscopy. Confocal images provide clear evidence of mitochondrial staining and additional ER localization. Confocal fluorescence images showed that the luminescence pattern is consistent with a mitochondrial distribution, with the merged images of red and green channels showing good correspondence. In addition, partial reticular staining has been observed on confocal images, which is suggestive of endoplasmic reticulum localization. All the compounds studied do not localize to the nucleus. The inspection of the fluorescence microscopy images showed ER–mitochondria localization at all incubation times, from 5 min to 48 h. No signs of rupture of plasma membrane have been observed and the cellular architecture and integrity of the mitochondria was preserved in the presence of the compounds. The images showed the typical pattern of mitochondrial organization present in MCF-7 and NHDF obtained in previous studies. Considering the nature of the chromophore structure on the compounds localization, no marked changes have been observed on their mitochondrial-ER distribution in the three different human cells. A possible explanation about their preferential accumulation in mitochondria is that at physiological as well as the mitochondrial membranes. Relatively to the additional reticular staining localization, this could be explained by spatial and functional organized network mediated by mitochondrial proteins and mitochondria-associated membranes that interconnect ER–mitochondria. In every case, all the compounds exhibited identical sub-cellular localization patterns on mitochondria and endoplasmic reticulum in less than 1 h. This result also indicates that the nature of the chromophore does not affect the cellular distribution. The fact that the compounds localize in both mitochondria and endoplasmic reticulum also makes them ideal candidates for their use as specific cellular probes, due to the intimate liaison of the endoplasmic reticulum–mitochondria. However, the compounds cell uptake mechanism requires future investigation. The timescale of trafficking of these compounds, the time-dependent localization and the cellular viability makes them promising molecular probes. Agonists and positive allosteric modulators of the a7 nicotinic acetylcholine receptor are currently being developed to ameliorate cognitive deficits in diseases such as schizophrenia, ADHD and Alzheimer’s disease. The a7 nAChR desensitizes rapidly in response to high agonist concentrations in vitro, which initially led to concern regarding its applicability as a clinical drug target.