Phasic and tonic smooth muscles express two myosin heavy chain isoforms that differ by only a seven amino acid insert in a flexible surface loop located near the nucleotide-binding site. The inserted isoform found predominantly in phasic muscle has two times the actin-activated ATPase activity and in vitro actin filament velocity as the non-insert isoform found mainly in tonic muscle (Kelley, C.A., Takahashi, M., Yu, J.H. & Adelstein, R.S. 1993. J Biol Chem 268, 12848, Rovner, A.S., Freyzon, Y. & Trybus, K.M. 1997. J Musc Res Cell Motil 18, 103). We used a laser trap to characterize the molecular mechanics of the inserted isoform [(+)insert] and of a mutant lacking the insert [(-)insert], which is analogous to the isoform found in tonic muscles. The constructs were expressed in the baculovirus/insect cell system. Unitary displacements (Duni) were similar for both the constructs (approximately 10 nm) but the attachment time (ton) for the (-)insert was two times that of the (+)insert. These data suggest that the insert in the nucleotide-binding loop does not affect the inherent mechanics of the myosin molecule but rather the kinetics of the cross-bridge cycle.