1. The common mechanism of action of non-steroidal anti-inflammatory drugs (NSAIDs) is the inhibition of the enzyme cyclo-oxygenase (COX), however, this inhibition is not enough to completely account for the efficacy of these agents in several models of acute pain. 2. It has been demonstrated that cholinergic agents can induce antinociception, but the nature of the interaction between these agents and NSAIDs drugs has not been studied. The present work evaluates, by isobolographic analysis, the interactions between the cholinergic indirect agonist neostigmine (NEO) and NSAIDs drugs, using a chemical algesiometric test. 3. Intraperitoneal (i.p.) or intrathecal (i.t.) administration of NEO and of the different NSAIDs produced dose-dependent antinociception in the acetic acid writhing test of the mouse. 4. The i.p. or i.t. co-administration of fixed ratios of ED(50) fractions of NSAIDs and NEO, resulted to be synergistic or supra-additive for the combinations ketoprofen (KETO) and NEO, paracetamol (PARA) and NEO) and diclofenac (DICLO) and NEO administered i.p. However, the same combinations administered i.t. were only additive. In addition, the combinations meloxicam (MELO) and NEO and piroxicam (PIRO) and NEO, administered either i.p. or i.t., were additive. 5. The results suggest that the co-administration of NEO with some NSAIDs (e.g. KETO, PARA or DICLO) resulted in a synergistic interaction, which may provide evidence of supraspinal antinociception modulation by the increased acetylcholine concentration in the synaptic cleft of cholinergic interneurons. The interaction obtained between neostigmine and the NSAIDs could carry important clinical implications.