MANY protein kinases are self-regulated by an intrasteric mechanism where part of the enzyme’s structure directly inhibits the active site(1,2). This inhibitory structure is called a pseudosubstrate and specific regulators are required to remove it from the active site to allow substrates access. Removal of the pseudosubstrate sequence from members of the myosin light-chain kinase subfamily(1), including twitchin kinase, activates them but it is not known whether the pseudosubtrate sequence binds to the active site. Native twitchin is a 753K protein (6,839 residues) located in muscle A-bands of the nematode Caenorhabditis elegans(3,4) and because of its size has not been easy to study. We have determined the crystal structure, refined to 2.8 Angstrom resolution, of a recombinant fragment (residues 5,890 to 6,262) of twitchin kinase(3,4) that contains the catalytic core and a 60 residue carboxy-terminal tail. The C-terminal tail extends through the active site, wedged between the small and large lobes of the structure and making extensive contacts with the catalytic core which accounts for autoinhibition and provides direct support for the intrasteric mechanism of protein kinase regulation.