The aim of this study was to develop an approach to directly assess protein fractional synthesis rate (FSR) in isolated human muscle fibers in a fiber type-specific fashion. Individual muscle fibers were isolated from biopsies of the vastus lateralis (VL) and soleus (SOL) obtained from eight young men during a primed, continuous infusion of [5,5,5-(2)H3]leucine performed under basal conditions. To determine mixed protein FSR, a portion of each fiber was used to identify fiber type, fibers of the same type were pooled, and the [5,5,5-(2)H3]leucine enrichment was determined via GC-MS. Processing isolated slow-twitch [myosin heavy chain (MHC) I] and fast-twitch (MHC IIa) fibers for mixed protein bound [5,5,5-(2)H3]leucine enrichment yielded mass ion chromatographic peaks that were similar in shape, abundance, and measurement reliability as tissue homogenates. In the VL, MHC I fibers exhibited a 33% faster (P<0.05) mixed protein FSR compared with MHC IIa fibers (0.068+/-0.006 vs. 0.051+/-0.003%/h). MHC I fibers from the SOL (0.060+/-0.005%/h) and MHC I fibers from the VL displayed similar (P>0.05) mixed protein FSR. Feasibility of processing isolated human muscle fibers for analysis of myofibrillar protein [5,5,5-(2)H3]leucine enrichment was also confirmed in non-fiber-typed pooled fibers from the VL. These methods can be applied to the study of fiber type-specific responses in human skeletal muscle. The need for this level of investigation is underscored by the different contributions of each fiber type to whole muscle function and the numerous distinct adaptive functional and metabolic changes in MHC I and MHC II fibers originating from the same muscle.