Previous studies on the mode of action of flavoxate have shown that the drug exerts a selective and direct muscle relaxant activity. In order to study the mode of action of flavoxate, the following activities were investigated: calcium blocking, inhibition of cyclic AMP phosphodiesterase (PDE), local anaesthetic activity, the effects on the synthesis and release of prostaglandins. In the K+-depolarized guinea-pig taenia coli, contracted by CaCl2, flavoxate and papaverine showed a moderate calcium antagonistic activity. Anticholinergic drugs, such as atropine and emepronium, did not exert a similar action. The antispasmodic activity of a drug can be correlated with inhibition of cyclic AMP phosphodiesterase, and since papaverine is a potent PDE inhibitor, we tested flavoxate for this activity. Flavoxate exerted a PDE inhibitory activity about three and five times greater than that of aminophylline in tissues homogenates of guinea-pig ureter and urinary bladder, respectively. It also showed the same local anaesthetic activity of lidocaine. Finally, the synthesis and release of prostaglandins by urinary bladder muscle in vitro have been investigated before and after treatment with flavoxate. Myolytic activity of papaverine and flavoxate do not involve inhibition of prostaglandins synthesis in rat urinary bladder in vitro. Therefore, the mode of action of flavoxate can be related to a superimposition of myotropic, calcium antagonistic and local anaesthetic activity.