The human norepinephrine (NE) transporter (hNET) attenuates neuronal signaling by rapid NE clearance from the synaptic cleft, and NET is a target for cocaine and amphetamines as well as therapeutics for depression, obsessive-compulsive disorder, and post-traumatic stress disorder. In spite its central importance in the nervous system, little is known about how NET substrates, such as NE, 1-methyl-4-tetrahydropyridinium (MPP⁺) or amphetamine, interact with NET at the molecular level. Nor do we understand the mechanisms behind transport rate. Previously we introduced a fluorescent substrate similar to MPP⁺, which allowed separate and simultaneous binding and transport measurement (1). Here we use this substrate, 4-(4-(dimethylamino)styrl)-N-methyl-pyridinium (ASP⁺), in combination with GFP-tagged hNETs to measure substrate-transporter stoichiometry and substrate binding kinetics. Calibrated confocal microscopy and fluorescence correlation spectroscopy (FCS) reveal that hNETs, which are homo-multimers, bind one substrate-molecule per transporter subunit. Substrate residence at the transporter, obtained from rapid on-off kinetics revealed in FCS, is 526 µsec. Substrate residence obtained by infinite dilution is 1000 times slower. This novel examination of substrate-transporter kinetics indicates that a single ASP⁺ molecule binds and unbinds thousands of times before being transported or ultimately dissociated from hNET. Calibrated fluorescent images combined with mass spectroscopy give a transport rate of 0.06 ASP⁺/hNET-protein/sec, thus 36,000 on-off binding events (and 36 actual departures) occur for one transport event. Therefore binding has a low probability of resulting in transport. We interpret these data to mean that inefficient binding could contribute to slow transport rates.