Determinations of fatigue ratio, twitch and tetanus tension, and contraction and half-relaxation times of the isometric twitch were made in 21 single fast-twitch motor units from the rat tibialis anterior muscle. Single motor units were functionally isolated by microdissection of the ventral root, and the glycogen depletion technique was used to demonstrate the muscle fibers in the unit. Morphological and immuno- and enzyme-histochemical methods were applied to serial muscle cross sections to characterize the muscle fibers in the unit. Three of the units had muscle fibers of the IIa type according to staining both for myofibrillar adenosinetriphosphatase after acid preincubation and with the use of monoclonal antibodies specific for myosin heavy chains (MHCs), i.e., the IIa-MHC isoform. The other 18 units were of the IIb type according to enzyme-histochemistry, but immunohistochemistry showed that in six of these units the muscle fibers exhibited the novel type IIx-MHC isoform and in the other 12 units the IIb-MHC isoform. It was found that the IIx motor units have contraction and half-relaxation times similar to those of types IIa and IIb units but have morphological, physiological, and biochemical properties that distinguish them from the latter two types.