Extraocular muscles are characterized by their faster rates of contraction and their higher resistance to fatigue relative to limb skeletal muscles. Another often reported characteristic of extraocular muscles is that they generate lower specific forces (sP(o), force per muscle cross-sectional area, kN/m(2)) than limb skeletal muscles. To investigate this perplexing issue, the isometric contractile properties of the levator palpebrae superioris (levator) and superior rectus muscles of the rat were examined in situ with nerve and blood supply intact. The extraocular muscles were attached to a force transducer, and the cranial nerves exposed for direct stimulation. After determination of optimal muscle length (L(o)) and stimulation voltage, a full frequency-force relationship was established for each muscle. Maximum isometric tetanic force (P(o)) for the levator and superior rectus muscles was 177 +/- 13 and 280 +/- 10 mN (mean +/- SE), respectively. For the calculation of specific force, a number of rat levator and superior rectus muscles were stored in a 20% nitric acid-based solution to isolate individual muscle fibers. Muscle fiber lengths (L(f)) were expressed as a percentage of overall muscle length, allowing a mean L(f) to L(o) ratio to be used in the estimation of muscle cross-sectional area. Mean L(f):L(o) was determined to be 0.38 for the levator muscle and 0.45 for the superior rectus muscle. The sP(o) for the rat levator and superior rectus muscles measured in situ was 275 and 280 kN/m(2), respectively. These values are within the range of sP(o) values commonly reported for rat skeletal muscles. Furthermore P(o) and sP(o) for the rat levator and superior rectus muscles measured in situ were significantly higher (P < 0.001) than P(o) and sP(o) for these muscles measured in vitro. The results indicate that the force output of intact extraocular muscles differs greatly depending on the mode of testing. Although in vitro evaluation of extraocular muscle contractility will continue to reveal important information about this group of understudied muscles, the lower sP(o) values of these preparations should be recognized as being significantly less than their true potential. We conclude that extraocular muscles are not intrinsically weaker than skeletal muscles.