The effect of amiloride on glycinergic transmission in mechanically dissociated rat spinal dorsal horn neurons was examined with the use of whole-cell patch-clamp recording. Amiloride reversibly reduced both the frequency and amplitude of spontaneous glycinergic miniature IPSCs (mIPSCs) and its inhibitory effect on glycinergic mIPSCs persisted in either Ca2+-free or Na+-free external solutions while it disappeared in K+-free external solution. Analysis of the relationship between mIPSCs amplitude and frequency at various holding potentials shows that amiloride inhibition of glycinergic mIPSC frequency could result indirectly from its inhibition of amplitude.