This work describes the relationship between the cytoplasmic free calcium concentration ([Ca2+]i) measured by the fluorescent Ca(2+)-indicator fura-2, the phosphorylation of the myosin light chain and the force development in the phasic longitudinal smooth muscle layer of guinea-pig ileum and the tonic rabbit pulmonary artery. The close temporal relationship between the rise in cytoplasmic Ca2+ and the initiation of force development as well as the rather good correlation between cytoplasmic Ca2+ and force maintenance leaves little doubt about cytoplasmic Ca2+ being the primary regulator of force. However the present experimental evidence indicate that [Ca2+]i and force are not invariably tightly coupled in smooth muscle. A dissociation between the time course of [Ca2+]i and force was found in the tonic rabbit pulmonary artery but not in the phasic ileum of the guinea-pig. In contrast, there was a pronounced decline in the Ca(2+)-sensitivity of the contractile apparatus (desensitization to Ca2+) in the guinea-pig ileum during prolonged depolarization, an observation not found in the pulmonary artery. Such desensitization could reflect the activation of highly active myosin light chain phosphatase(s) and the different Ca(2+)-sensitivities of tonic and phasic smooth muscles can, at least in part, be due to differences in myosin light chain kinase/phosphatase activity ratios. The sensitivity of the regulatory/contractile apparatus to Ca2+ was increased by agonists in intact and in permeabilized preparations. Furthermore a different sensitizing potentiation between different agonists was observed. The mechanism of the "sensitization" of the contractile response to Ca2+ could act through the activation of the phosphorylation of a protein phosphatase inhibitor, thereby inhibiting the myosin light chain phosphatase. The experiments therefore show that different levels of tension may be present at the same [Ca2+]i and indicate that the Ca(2+)-sensitivity can be modulated in smooth muscle.