Necessity of newly synthesized ATP by creatine kinase for synthesis of ATP as an energy source for smooth muscle contraction was studied in permeabilized longitudinal muscle preparations of rat proximal colon. In alpha-toxin-permeabilized preparations, Ca++ induced "phasic type" contraction in a normal bath solution containing 4 mM ATP and 5 mM phosphocreatine. Omission of phosphocreatine from the solution resulted in significant decrease in phasic contraction, and omission of ATP resulted in loss of the response to Ca++. When ADP, but not adenosine-5-O-(2-thiodiphosphate), with phosphocreatine was added as a substitute for ATP, Ca++ induced the same type of contraction as with ATP. The maximum tensions of the phasic and tonic phases of the contraction with ADP were approximately 60% of, and almost the same, respectively as those with ATP. A selective inhibitor of creatine kinase, 2,4-dinitrofluorobenzene, inhibited the phasic contraction induced by Ca++. After irreversible inhibition of endogenous creatine kinase by DNFB in beta-escin-permeabilized preparations, treatment of the preparations with exogenous creatine kinase restored Ca(++)-induced contraction. These findings suggest that ATP synthesized from ADP and phosphocreatine by creatine kinase was necessary for phasic contraction of permeabilized smooth muscle and that exogenous ATP was mainly used after its hydrolysis to ADP.