Two smooth muscle myosin heavy chain isoforms differ by a 7-amino- acid insert in a flexible surface loop located near the nucleotide binding site. The non-inserted isoform is predominantly found in tonic muscle, while the inserted isoform is mainly found in phasic muscle. The inserted isoform has twice the actin-activated ATPase activity and actin filament velocity in the in vitro motility assay as the non-inserted isoform. We used the laser trap to characterize the molecular mechanics and kinetics of the inserted isoform ((+)insert) and of a mutant lacking the insert ((−)insert), analogous to the isoform found in tonic muscle. The constructs were expressed as heavy meromyosin using the baculovirus/insect cell system. Unitary displacement (d) was similar for both constructs (∼10nm) but the attachment time (ton for the (−)insert was twice as long as for the (+)insert regardless of the [MgATP]. Both the relative average isometric force (Favg(−insert)/Favg(+insert))=1.1±0.2 (mean±se) using the in vitro motility mixture assay, and the unitary force (F∼1pN) using the laser trap, showed no difference between the two constructs. However, as under unloaded conditions, ton under loaded conditions was longer for the (−)insert compared with the (+)insert construct at limiting [MgATP]. These data suggest that the insert in this surface loop does not affect the mechanics but rather the kinetics of the cross-bridge cycle. Through comparisons of ton from d measurements at various [MgATP], we conclude that the insert affects two specific steps in the cross-bridge cycle, that is, MgADP release and MgATP binding.