Live cell imaging represents an important technique in the study of biological processes; the use of fluorescent proteins or dyes provides an important tool for the in vivo visualization, in space, and time, of virtually any cellular mechanism or structure, avoiding artifacts or sample alteration that could occur with fixation methods. In this study we show that NRB-AF12 is endowed with features that make this dye an eligible prototype of new fluorescent probes, alternatives to ER-Tr, for live cell imaging (Figure 10). Furthermore, in NRB-sensitive single rat vascular myocytes, this dye, due to its constriction effect (manuscript in preparation), could also be employed to study the morphologic changes that occur in labeled intracellular organelles, as well as in the plasma membrane, during the contractile process. Whatever the binding site/s for NRB-AF12, this dye shows a cellular distribution near identical to that of ER-Tr in real-time fluorescent imaging using different cultured cell lines and freshly isolated rat caudal artery myocytes. NRB-AF12 labeling was also observed in fixed and permeabilized cells, allowing its use in immunocytochemistry co-localization experiments. In all cells tested, NRB-AF12 stained not only ER, but also other subcellular compartments such as Golgi apparatus, mitochondria, and organelles involved in the endocytic pathway (i.e., endosomes and lysosomes). NRB-AF12 internalization rate was extremely fast, since cell incubation with the fluorescent compound resulted in a well-defined meshwork staining of the cellular structures within a few minutes, and could be observed up to 3 h following its removal from the culture medium (Figure 11A). Another useful feature is that NRB-AF12 exhibited no apparent cytotoxicity, a serious disadvantage that often occurs using fluorescent probes for live cell imaging. Cell viability experiments demonstrated the dye to be biocompatible, having no harmful effects in the different cell lines (both human and rat) after 24-h incubation, even at concentrations (10 μM) much higher than those (500 ηM) needed to visualize cellular organelles (Figures 11B–D). Furthermore, the lack of cytotoxicity of NRB-AF12 allows the same sample to be repeatedly stained with the dye (for example at different time points within an experiment), without morphological alteration of cell structures or cell death; concurrently, the ability of NRB to effectively label its cellular target is preserved on re-exposure to the dye (Figure 11A).