Abstract Inhibitors of adenosine kinase, but not adenosine deaminase, produce antinociception when administered spinally. In this study, we evaluated the relative contribution of adenosine kinase and adenosine deaminase to the regulation of adenosine release into the extracellular space within the spinal cord by determining the effects of the adenosine kinase inhibitors 5−amino-5−deoxyadenosine and 5-iodotubercidin, and the adenosine deaminase inhibitor 2−deoxycoformycin on adenosine release from spinal cord slices in an in vitro perfusion system. Both 5−amino-5−deoxyadenosine (5–50,μM) and 5-iodotubercidin (5–50 μM), but not 2−deoxycoformycin (50 μM), augmented adenosine release. 5-Iodotubercidin was slightly more potent and effective than 5−amino-5−deoxyadenosine in augmenting release except at the highest concentration, where it was considerably more effective. Combinations of 2−deoxycoformycin (50 μM) and minimally active concentrations of 5−amino-5−deoxyadenosine and 5-iodotubercidin (5 μM each) produced a synergistic enhancement of release. These results support a predominant involvement of adenosine kinase in regulating extracellular adenosine levels in the spinal cord, but adenosine deaminase also can play a significant role.