WE investigated whether glycinergic transmission develops organotypically in auditory brain stem cultures. Slices of the medial nucleus of the trapezoid body and the lateral superior olive were incubated in medium with a raised extracellular K+concentration. Asin vivo, glycine receptor α1 subunit immunoreactivity increased and became clustered on somata and proximal dendrites. Together with organotypic expression of glycine transporter GLYT2, this indicates that molecular components of glycinergic synapses form properly. In contrast, glycinergic synaptic currents did not develop asin vivo: after 7 daysin vitrothey were still similar to those at the time of culture preparation. We suggest that for organotypic development of glycine receptors and transporters, Ca2+influx due to elevated K+is sufficient. The development of functional synaptic transmission, however, may require patterned electrical activity.