The chronic effects of glycine transporter 1 and 2 inhibitors (sarcosine and ALX-1393, respectively) on miniature inhibitory postsynaptic currents were studied in cultured spinal neurons. We found that sarcosine increased the frequency of overall miniature inhibitory postsynaptic currents without affecting the ratio of glycinergic, mixed and GABAergic miniature inhibitory postsynaptic currents, whereas ALX-1393 changed the ratio by increasing the proportions of GABAergic and mixed miniature inhibitory postsynaptic currents without affecting overall mIPSC frequency. We propose that inhibition of glycine transporter 1 by sarcosine increased overall mIPSC frequency via the activation of presynaptic glycine receptors, while inhibition of glycine transporter 2 by ALX-1393 changed the ratio of glycinergic, mixed and GABAergic miniature inhibitory postsynaptic currents by shifting the balance of inhibitory transmitters in vesicles towards gamma-aminobutyric acid.