Although co-transmission from many autonomic neurons undoubtedly takes place, it is unclear whether neurotransmitter storage takes place within the same or separate vesicles. The use of fluorescent neurotransmitter transporter substrates allows monitoring of vesicular uptake (Parker et al., 2010), indicating the presence of specific vesicles, and may allow the question of co-storage of purines and catecholamines to be definitively answered. Cultured PC-12 cells were exposed to a fluorescent catecholamine transporter and vesicular monoamine transporter (VMAT) substrate (Neurotransmitter Transporter Uptake Assay; NTUA) using protocols similar to those described by Parker et al. (2010) or the SLC17A9 substrate quinacrine. SLC17A9 has recently been identified as the vesicular ATP transporter, and has hence been called the vesicular nucleotide uptake transporter (VNUT)(Sawada et al., 2008). Fluorescence accumulation was monitored in real time using fluorescence microscopy. NTUA was excited at 405nm; quinacrine was excited at 456nm. Quinacrine (1 μM) was found to accumulate in a punctuate manner in PC12 cells, (12.4 ± 3.1 granules per cell; mean ± S.E.M.), as was NTUA (1:100) to a similar extent (9.5 ± 1.8 granules per cell; Student’s t-test vs quinacrine, p>0.05, 24 cells, n=4). Accumulation of quinacrine was found to be sensitive to the SLC17A9 inhibitor Evans blue (1 μM; 1.3 ± 0.3 granules per cell; p<0.05, 24 cells, n=4). NTUA accumulation was insensitive to the noradrenaline transporter inhibitor desipramine (1 µM; 8.4 ± 1.2 granules per cell; p>0.05, 22 cells, n=4), but was sensitive to the dopamine transporter inhibitor GBR 12909 (100 nM; 4.7 ± 0.4 granules per cell; p<0.05, 24 cells n=4). In this investigation, two fluorescent neurotransmitter transporter substrates are evaluated for their specificity. A similar number of vesicles were detected using NTUA and quinacrine, suggesting co-localization or a similar number of granules for ATP and catecholamines. Co-localization experiments, requiring careful spectral separation or novel experimental approaches, may indicate whether or not ATP can be co-stored in vesicles with catecholamines. Altered neurotransmitter release can take place in healthy ageing, and may be a causative factor in a range of CNS, and PNS pathologies (Burnstock, 2007). Targeted modulation of vesicular co-storage may in future offer an alternative therapeutic approach.