This study evaluated the pain enhancing properties of the adenosine A3 receptor agonist N6-benzyl-5'-N-ethylcarboxamidoadenosine (N6-benzyl-NECA) by assessing behavioural effects following s.c. administration alone to the dorsal hindpaw of the rat, or in combination with a low concentration of formalin (0.5%). Edema formation was monitored by determining paw volume with plethysmometry. N6-benzyl-NECA (0.005-10 nmol) produced a dose-related increase in intrinsic flinching behaviours, as well as an increase in phase 2A flinch responses in the presence of formalin. Intrinsic effects were blocked by the histamine H1 receptor antagonist mepyramine and the 5-hydroxytryptamine2 (5-HT2) receptor antagonist ketanserin, but not by other 5-HT receptor antagonists or adenosine A1 or A2 receptor antagonists. N6-benzyl-NECA also produced an increase in paw volume, both alone and in the presence of formalin, with higher doses being required to produce this effect than for the flinch response. The increase in paw volume was also blocked by mepyramine and ketanserin but not by other antagonists. These results indicate both a nociceptive response and a proinflammatory response resulting in edema formation following activation of adenosine A3 receptors which is mediated by both 5-HT and histamine released most likely from mast cells.