The mechanism responsible for the regulation of smooth muscle tone at low levels of myosin light chain (MLC) phosphorylation is still poorly understood. According to one model, so-called latchbridges, which contribute to force maintenance at low levels of MLC phosphorylation, are generated by dephosphorylation of attached and phosphorylated crossbridges. The model predicts that the force generated for a given level of MLC phosphorylation depends on the activity of the MLC phosphatase. We tested this hypothesis by reducing the activity of the phosphatase by at least 80% in two ways: inhibition with okadaic acid and extraction. Under both conditions, higher levels of MLC phosphorylation were required to support a given level of force, suggesting a decreased flux of attached phosphorylated to attached dephosphorylated crossbridges, as predicted by this model. Although, under both conditions, the relationship between force and MLC phosphorylation was shifted to the right, the curves did not superimpose as would have been expected if the phosphatase activity were the only determinant of the coupling between force and phosphorylation. In the extracted fibres, two more proteins, calponin and SM22, were significantly reduced in addition. Therefore, these proteins might be involved in modulating the relationship between force and MLC phosphorylation.