Myosin heavy chain (MHC) isoforms in vertebrate striated muscles are distinguished functionally by differences in chemomechanical kinetics. These kinetic differences may influence the cross-bridge dependent cooperativity of thin filament Ca²⁺-activation. To determine if Ca²⁺-sensitivity of unloaded thin filament sliding depends upon MHC isoform kinetics, we performed in vitro motility assays with rabbit skeletal heavy meromyosin (rsHMM) or porcine cardiac myosin (pcMyosin). Regulated thin filaments were reconstituted with recombinant human cardiac troponin (rhcTn) and & [alpha]-Tm (rhcTm) expressed in E. coli. All three subunits of rhcTn were co-expressed as a functional complex using a novel construct with a GST affinity tag at the N-terminus of hcTnT and an intervening tobacco etch virus (TEV) protease site that allows purification of rhcTn without denaturation, and removal of the GST tag without proteolysis of hcTn subunits. Use of this highly purified rhcTn in our motility studies resulted in a clear definition of the regulated motility profile for both fast and slow MHC isoforms. Maximum sliding speed (pCa 5) of regulated thin filaments was ~ 5-fold faster with rsHMM compared with pcMyosin, although speed was increased by 1.6 - 1.9- fold for regulated over unregulated actin with both MHC isoforms. The Ca²⁺-sensitivity of regulated thin filament sliding speed was unaffected by MHC isoform. Our motility results suggest that the cellular changes in isoform expression that result in regulation of myosin kinetics can occur independently of changes that influence thin filament Ca²⁺-sensitivity.