OBJECTIVE To investigate the distribution of the sarcoplasmic reticulum fast-twitch Ca(2+)-pump (Ca(2+)-adenosine triphosphatase [Ca(2+)-ATPase]) in extraocular muscle fiber populations of the rat and rabbit, using fast Ca(2+)-adenosine triphosphatase-specific monoclonal antibodies. METHODS Adult female rats and rabbits were killed with overdose of sodium pentobarbital, and the extraocular muscles were rapidly frozen in isopentane cooled with liquid nitrogen. Serial transverse frozen sections were cut and labeled respectively with monoclonal antibodies specific for various isoforms of myosin heavy chain, and monoclonal antibodies specific for the fast isoform of the sarcoplasmic reticulum Ca(2+)-ATPase. CONCLUSIONS In rat extraocular muscle, fast type 2 myosin heavy chain and fast Ca(2+)-ATPase are coexpressed in both singly- and multiply-innervated fibers of the orbital layer near the endplate band, but distally both proteins drop out entirely, indicating that the contraction and relaxation properties of orbital fibers vary along their length. Also, fast Ca(2+)-ATPase persists more distally in orbital fibers than does type 2 myosin heavy chain, suggesting that their joint expression is not directly coregulated. This conclusion is reinforced in rabbit, whose orbital multiply-innervated fibers express fast Ca(2+)-ATPase but do not express type 2 myosin heavy chain at all. Fast Ca(2+)-ATPase is expressed in all global layer fast-twitch singly-innervated fibers, but not in global multiply-innervated fibers. In light of known differences in myosin heavy chain expression, the two populations of multiply-innervated fibers must differ with respect to both contractility and relaxation.