Several lines of evidence indicates that endogenous enkephalinergic system is involved in antinociceptive response in the spinal cord. However enkephalins are rapidly broken-down by endogenous enzymes, and therefore enkephalin-degradation enzyme inhibitors are considered as potential analgesics. Purinergic agonists adenosine triphosphate (ATP), a non- selective agonist for several ionotropic P2X and metabotropic P2Y receptor subtypes, function as peripheral pain mediator through mechanism involving increased sensitivity of peripheral nociceptive sensory fibres. By using intracellular calcium as a key nociceptive signal we investigated the effects of two endogenous enkephalinase inhibitors, opiorphin and spinorphin, on ATP-induced increase of intracellular calcium in cultured rat dorsal root ganglion (DRG) neurons. Following enzymatic digestion and mechanical agitation the DRG neurons were cultured on coated coverslips and loaded with 5 µM Fura-2 AM. Standard fura-2 ratiometric technique was utilised for quantifying [Ca2+]i responses in individual DRG neurons using fluorescence imaging system consisting of CCD camera coupled to an inverted microscope. For each experiment cells were consecutively stimulated twice with consistent duration of ATP application. Effects of opiorphin and spinorphin were tested on response to the second stimulus with ATP. To avoid variations in the [Ca2+]i responses to the stimulation with ATP across experiments, the responses were normalized. All data were analyzed by using unpaired t test, P <.05 defining statistical significance. The rise in [Ca2+]i in response to ATP was significantly smaller after its second application in the presence of opiorphin than its first control application [(100±0% (n=16, basal response to ATP) vs. 54.7±4.4% (n=16, P>0.001 for application of ATP with 10 µM opiorphin); and 100±0% (n=16, basal response to ATP) vs. 5.2±3.9% (n=70, P>0.0001 for application of ATP in the presence of 100 µM opiorphin), respectively]. Application of spinorphin also attenuated Ca2+ responses to ATP in a dose dependent manner [(100±0% (n=46, basal ATP response) vs. 97.9±4.4% (n=46, P<0.05) for application of ATP with 10 µM spinorphin; and 100±0% (n=51, basal response to ATP) vs. 58.6±4.8% (n=51, P>0.001) for application of ATP in the presence of 100 µM spinorphin, and 100±0% (n=47, basal response to ATP) vs. 58.6±4.8% (n=47, P>0.0001) for application of ATP in the presence of 300 µM spinorphin, respectively]. In conclusion the study demonstrate that enkephalinase inhibitors, opiorphin and spinorphin, attenuates ATP-induced increase of intracellular calcium in cultured rat DRG neurons.