In this work, we show that beta-carbolines, which are known negative allosteric modulators of GABA(A) receptors, inhibit glycine-induced currents of embryonic mouse spinal cord and hippocampal neurons. In both cell types, beta-carboline-induced inhibition of glycine receptor (GlyR)-mediated responses decreases with time in culture. Single-channel recordings show that the major conductance levels of GlyR unitary currents shifts from high levels (> or = 50 pS) in 2 to 3 days in vitro (DIV) neurons to low levels (<50 pS) in 11 to 14 DIV neurons, assessing the replacement of functional homomeric GlyR by heteromeric GlyR. In cultured spinal cord neurons, the disappearance of beta-carboline inhibition of glycine responses and high conductance levels is almost complete in mature neurons, whereas a weaker decrease in beta-carboline-evoked glycine response inhibition and high conductance level proportion is observed in hippocampal neurons. To confirm the hypothesis that the decreased sensitivity of GlyR to beta-carbolines depends on beta subunit expression, Chinese hamster ovary cells were permanently transfected either with GlyR alpha2 subunit alone or in combination with GlyR beta subunit. Single-channel recordings revealed that the major conductance levels shifted from high levels (> or = 50 pS) in GlyR-alpha2-transfected cells to low levels (<50 pS) in GlyR-alpha2+beta-containing cells. Consistently, both picrotoxin- and beta-carboline-induced inhibition of glycine-gated currents were significantly decreased in GlyR-alpha2+beta-transfected cells compared with GlyR-alpha2-containing cells. In summary, we demonstrate that the incorporation of beta subunits in GlyRs confers resistance not only to picrotoxin but also to beta-carboline-induced inhibition. Furthermore, we also provide evidence that hippocampal neurons undergo in vitro a partial maturation process of their GlyR-mediated responses.