The circular dichroic (CD) and fluorescent spectral properties of the myosin head (subfragment I (SFI)) modified by covalently bridging the 2 essential thiol groups were examined. CD spectra of SFI with the 2 thiols linked through reaction with a bifunctional reagent, N,N''-p-phenylenedimaleimide, show enhancement of the 282-nm minimum similar to that observed for the long-lived kinetic intermediate (Mg**MgADP.cntdot.Pi) formed during the ATP cleavage reaction. No significant perturbation of the CD band at 282 nm is seen on blocking both thiol groups with the monofunctional reagent N-ethylmaleimide. The fluorescence emission maximum also shifts to lower wavelengths following covalent bridging (343-340 nm), but no change in fluorescent intensity was detected. Formation of the covalent bridge completely inhibits interaction of the modified protein with F-actin. The local conformational state of the polypeptide chain formed on bridging the 2 thiol groups exhibits certain similarities with the state produced following binding of MgATP to native myosin.