Mechanical responses to noradrenaline (NA) were investigated in the rat vas deferens exposed to Ca-free solution containing 0.5 mM-EGTA. A tonic response was produced in Ca-free solution at the epididymal portion, while almost no response could be observed at the prostatic portion. In most experiments NA (10(-4) M) was applied for 4 min, every 20 min. The absolute tension development in Ca-free solution was usually 60-80% of the control tonic response in the presence of 2.4 mM-Ca. The response could be produced repeatedly, even after exposure to Ca-free solution for more than 20 hr, without a significant decrease. During the first hour of exposure to Ca-free solution, the rate of rise and the magnitude of the NA contraction increased and then remained constant, though the relaxation became slow. Transient treatment with 2.4 mM-Ca slightly suppressed the subsequent NA response in Ca-free solution. Similarly, the NA response was smaller during readmission of 0.2-0.5 mM-Ca than that obtained before Ca readmission. A high concentration of verapamil (2 X 10(-4) M) reversibly reduced the NA response by about 70% after 30 min. Theophylline (10 mM) and dibutyryl cyclic AMP (10(-4) M) also reversibly suppressed the NA response, the suppression being about 80%. None of these substances produced a tension change by themselves. The suppressing effect may be mediated via an increase of intracellular cyclic AMP which reduces phosphorylation of myosin. Caffeine (10 mM) and dibutyryl cyclic GMP (10(-4) M) had similar but much weaker effects than theophylline and dibutyryl cyclic AMP. A calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide (W-7) slowly reduced the NA response. The block was nearly complete after 30 min treatment with 3 X 10(-4) M-W-7, and the recovery was very poor after prolonged exposure. This effect of W-7, which is the same in the presence and absence of Ca, suggests that a Ca-calmodulin reaction is involved in the NA response in Ca-free solution. Fluoride at a concentration higher than 3 mM increased the muscle tone in the absence of external Ca, and transiently potentiated the NA response. In the presence of F-, the relaxation of the NA response was incomplete and the muscle tone increased stepwise after each NA application. When the muscle tone became higher than the NA response in the absence of F-, the NA response was abolished. The action of several metabolic inhibitors (2,4-dinitrophenol, carbonylcyanide chlorophenyl hydrazone, NaCN, monoiodoacetate) was similar to that of F-, suggesting that they release Ca from mitochondria, causing tension development. The observations are consistent with the hypothesis that the contraction of the vas deferens caused by NA in the absence of external Ca depends on the availability of intracellular Ca, stored in mitochondria and released by NA.