The effect of two positive modulators, RE1 and EX15, on the voltage-gated K⁺ channel K_v3.1 was investigated using the whole-cell patch-clamp technique on HEK293 cells expressing K_v3.1a. RE1 and EX15 increased the K_v3.1 currents in a concentration-dependent manner with an EC₅₀ value of 4.5 and 1.3µM, respectively. However, high compound concentrations caused an inhibition of the K_v3.1 current. The compound-induced activation of K_v3.1 channels showed a profound hyperpolarized shift in activation kinetics. 30µM RE1 shifted V_½ from 5.63±0.31mV to -9.71±1.00mV and 10µM EX15 induced a shift from 10.77±0.32mV to -15.11±1.57mV. The activation time constant (Tau_act) was reduced for both RE1 and EX15, with RE1 being the fastest activator. The deactivation time constant (Tau_deact) was also markedly reduced for both RE1 and EX15, with EX15 inducing the most prominent effect. Furthermore, subjected to depolarizing pulses at 30Hz, both compounds was showing a use-dependent effect resulting in a reduction of the compound-mediated effect. However, during these conditions, RE1- and EX15-modified current amplitudes still exceeded the control condition amplitudes by up to 200%. In summary, the present study introduces the first detailed biophysical characterization of two new K_v3.1 channel modifying compounds with different modulating properties.