1. The ATP action on spontaneous miniature glycinergic inhibitory postsynaptic currents (mIPSCs) was investigated in rat substantia gelatinosa (SG) neurons mechanically dissociated from the 2nd layer of the dorsal horn in which their presynaptic glycinergic nerve terminals remained intact. 2. ATP reversibly facilitated the frequency of the mIPSCs in a concentration-dependent manner without affecting their amplitude distribution. The ATP agonist, 2-methylthioATP (2MeSATP), mimicked the ATP action, while another ATP receptor agonist, alphabeta-methylene-ATP (alpha,beta-meATP), had no effect on mIPSCs. 3. The ATP receptor antagonists, suramin (1 x 10-6 M) and pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (1 x 10-5 M), completely blocked the facilitatory effect of ATP on glycine release (102.0 +/- 11.2 % and 99.3 +/- 16.2 %, n = 6, respectively) without altering the current amplitude distributions. 4. N-Ethylmaleimide (NEM), a sulphydryl alkylating agent, suppressed the inhibitory effect of adenosine on mIPSC frequency (111.2 +/- 13. 3 %, n = 4) without altering the current amplitude distribution. However, ATP still facilitated the mIPSC frequency (693.3 +/- 245.2 %, n = 4) even in the presence of NEM. 5. The facilitatory effect of ATP (1 x 10-5 M) on mIPSC frequency was not affected by adding 1 x 10-4 M Cd2+ to normal external solution but was eliminated in a Ca2+-free external solution. 6. These results suggest that ATP enhances glycine release from nerve terminals, presumably resulting in the inhibition of SG neurons which conduct nociceptive signals to the CNS. This presynaptic P2X-type ATP receptor may function to prevent excess excitability in SG neurons, thus preventing an excessive pain signal and/or SG cell death.