Acetylsalicylic acid and morphine are the most widely distributed and most frequently used drugs in the relief of pain, but their analgesic activity has adverse side-effects. Mixtures containing these two drugs are frequently used to relieve mild to moderate pain despite the paucity of relevant experimental evidence so far published. We set out to study the possible antinociceptive effect of a combination of subactive doses of the two drugs in rats. A combination of low doses of acetylsalicylic acid (50 mg/kg i.p.) and morphine (3 mg/kg s.c.) was administered and the pain threshold was evaluated in the hot-plate and formalin tests, and 5-HT2 receptor binding capacity, 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) levels were measured in the cortex and pontine areas of the brain. The combination of acetylsalicylic acid and morphine had an analgesic effect in both tests that was associated with an increase in 5-HT levels and a decrease in 5-HT2 receptors in the cortex. These effects were either completely abolished or partially prevented by i.p. pretreatment with naloxone (1 mg/kg i.p.). Our results demonstrate that subactive doses of acetylsalicylic acid and morphine can exert analgesic and biochemical effects when given in combination in the rat and suggest an involvement of serotonergic and opiatergic systems.