In the double mannitol gap arrangement the contraction was estimated in single fibres isolated from rat extensor digitorum longus (e.d.l.) muscles that had been denervated for 2-48 days. Denervation induced large changes in the characteristics of the action potential and of the twitch. Up to 15-20 days after denervation the contraction-depolarization curve was sigmoidal and the maximum amplitude of the contraction was not modified by variation of [Ca]o or [Na]o. After 15-20 days of denervation a bell-shaped curve described the relation between contraction and potential. The maximum amplitude was dependent upon the [Ca]o. In Ca-free solution no contractile response was obtained. In Na-free, Ca-containing solution the relationship between contraction and potential was not modified by the addition of divalent cations Co2+, Cd2+, Mn2+, Mg2+, Ni2+, or Ba2+. The contraction, which appeared in Ca-free solution, was restored by adding Sr2+. D600, verapamil and bepridil failed to change the amplitude of the contraction while a marked reduction was found with dihydropyridines. The reduction was overcome by increasing [Ca]o. The present results suggest that the slow calcium current is not involved in the generation of the contractile responses developed by denervated muscles in Na-free (TEA) solution.