"Catch" is the state where some invertebrate muscles sustain high tension for long periods at low ATP hydrolysis rates. Physiological studies using muscle fibers have not yet fully provided the details of the initiation process of the catch state. The process was extensively studied by using an in vitro reconstitution assay with several phosphatase inhibitors. Actin filaments bound to thick filaments pretreated with the soluble protein fraction of muscle homogenate and Ca 2؉ (catch treatment) in the presence of MgATP at a low free Ca 2؉ concentration (the catch state). Catch treatment with >50 M okadaic acid, >1 M microcystin LR, 1 M cyclosporin A, 1 M FK506, or 0.2 mM calcineurin autoinhibitory peptide fragment produced almost no binding of the actin filaments, indicating protein phosphatase 2B (PP2B) was involved. Use of bovine calcineurin (PP2B) and its activator calmodulin instead of the soluble protein fraction initiated the catch state, indicating that only PP2B and calmodulin in the soluble protein fraction are essential for the initiation process. The initiation was reproduced with purified actin, myosin, twitchin, PP2B, and calmodulin. 32 P autoradiography showed that only twitchin was dephosphorylated during the catch treatment with either the soluble protein fraction or bovine calcineurin and calmodulin. These results indicate that PP2B directly dephosphorylates twitchin and initiates the catch state and that no other component is required for the initiation process of the catch state.Invertebrate muscles such as the smooth adductor and byssus retractor muscles of bivalves maintain high tension with little energy expenditure after active contractions (1, 2). This high tension state, called the "catch state," is terminated by a neurotransmitter, 5-hydroxytryptamine (3), activating cAMPdependent protein kinase (PKA) 1 through an increase in the intracellular concentration of cAMP. When a catalytic subunit of PKA was added to chemically demembranated catch muscles, the passive tension of the catch state diminished (4). Phosphorylation of a target protein(s) by this kinase thus causes relaxation of the catch tension.Twitchin is a candidate for this target protein. Originally identified in the nematode Caenorhabditis elegans (5, 6), it is also called "mini-titin" and is abundantly present in molluscan muscles (7,8). Studies with the anterior byssus retractor muscle of the mussel suggested that its phosphorylation by PKA was involved in the relaxation of the catch state (9). The catalytic subunit of PKA phosphorylates 2-3 serine residues of the mussel catch muscle twitchin (10, 11). In contrast to the relaxation of the catch state, the regulation of the reverse reaction, i.e. initiation of the catch state, has not been well characterized yet, since the reaction in vivo seems to occur in parallel with the activation of muscle contraction. Therefore, how initiation signals are relayed onward and amplified in cascades and which types of phosphatases dephosphorylate twitchin to initiate the catch state are stil...