Characteristics of supersensitivity induced by the pretreatment with AF64A, an inhibitor of choline uptake at parasympathetic nerve endings, were examined in rat iris sphincter. In preparations Isolated and skinned by β-escin after the micro-injection of AF64A to eyes in vivo, the amplitude of maximum contraction in pCa 4.5 solution was increased by 180% of the control from the contralateral eyes. The Ca²⁺ sensitivity of the contractile system was slightly but significantly increased by AF64A injection; the half maximum contraction was obtained at pCa 5.87 and 6.05 in the control and AF64A-injected eyes, respectively. The increase in maximum contraction in AF64A-injected ones was neither affected by the addition of calmodulin, GTPγS nor H-7. The increase in Ca²⁺ sensitivity by AF64A injection was not affected by calmodulin, enhanced by GTPγS and abolished by H-7. AF64A injection increased the total protein content only by 30% of the control. The contents of contractile proteins per iris were quantified using Western blotting with monoclonal antibodies. The contents of actin and calponin were increased by AF64A, whereas those of myosin, calmodulin and caldesmon were not affected. The results indicate that AF64A-induced enhancement of the maximum contraction is not mainly due to the increase in the contents of major contractile proteins and that the increase in Ca²⁺ sensitivity could be due to the mechanism in which changes in protein kinase C and/or GTP binding protein activity are involved.