Aims: Excitatory Amino Acid Transporters (EAATs) are crucial for maintaining low extracellular glutamate concentrations in the mammalian brain. Apart from mediating secondary-active glutamate transport, EAATs also function as anion channels, however the molecular basis of anion conduction is insufficiently understood. Recent data suggested that mutations in the second transmembrane domain (TM2) alter anion permeation properties. We studied the effects of two mutations within TM2, S74V and D83A, on time- and voltage-dependent changes of M205C EAAT3 current amplitudes and fluorescence intensities by voltage-clamp fluorometry (VCF). Methods: We employed voltage-clamp fluorometry on the neuronal glutamate transporter hEAAT3 heterologously expressed in Xenopus laevis oocytes. The transporter was labeled at position C205 with AlexaFluor546. Additionally two mutations were inserted, S74V and D83A. Oocytes were incubated for 3-6 days after RNA injection and labeled directly before electrophysiological and fluorescence measurements. Results: VCF reveals voltage- and substrate-dependent fluorescence changes in the environment of AlexaFluor546-labeled C205 in EAAT3 (max. DF_NaCl [%] = 14.4 ± 0.5). Voltage dependence of fluoresence intensities is modified by application of Na⁺ and/or glutamate and upon modification of external anions. However, fluorescence changes occur upon changes of membrane potential also in the absence of Na⁺ as well as of glutamate. These changes are abolished by application of the transporter blocker TBOA, indicating that conformational changes are linked to movements of hairpin 2 (HP2). S74V and D83A modify anion channel function and affect the voltage, substrate and anion dependence of fluorescence changes. Conclusion: Our results indicate that fluorescence changes in the environment of C205 in EAAT3 are partly due to movements of HP2. They report conformational changes that are not part of glutamate transport cycle and that are modified by anion binding.