The α3-containing glycine channels (GlyR) are found in discrete areas of the spinal cord and hippocampus, but despite their likely physiological relevance, their kinetic properties are unknown. We investigated the activation mechanism of recombinant α3 rat homomeric glycine receptors. Cell-attached steady-state single channel recordings were obtained at 50 - 10000 μM glycine. Macroscopic synaptic-like glycine-evoked currents were obtained by applications of pulses of glycine (1 ms, 10 mM) to outside-out patches (intracellular chloride concentration 20 mM). Kinetic mechanisms were tested using maximum likelihood fits by the HJCFIT program to sets of idealized single channel records. The adequacy of each mechanism was judged by comparing the predictions of the model with the summary statistics of the single channel data and the time course of macroscopic deactivation.The single channel open probability of homomeric α3 GlyR was strongly concentration-dependent, with a Hill slope of of 3.7 ± 0.1, much steeper than that of α GlyRs (1.82 ± 0.24, Beato et al., 2004). This suggests that α3 GlyR require all five binding sites to bind glycine in order to reach their maximum open probability. In other homomeric Cys-loop channels, including α GlyR, occupancy of three out of five sites is sufficient.Other features of a3 GlyR activation were similar to those of other GlyR. In particular, the fully-liganded opening rate constant was 150,000 ± 24,000 s-1 and the overall efficacy was 67 ± 4. The microscopic affinity of glycine for the intermediate shut “flip” conformation was 160 ± 24 μM, approximately 5-fold higher than for the resting conformation (890 ± 80 μM; n = 3 sets). This accounted for the apparent cooperativity of the response.Beato, Groot-Kormelink, Colquhoun & Sivilotti (2004). J Neurosci 24, 895.