The proteolytic susceptibility of the subfragment 2/light meromyosin junction [heavy meromyosin (HMM) junction] of myosin was employed as a probe of the crossbridge conformation. The proteolysis was carried out in the myofibrils where myosin assembled in arrays typical of the in vivo organization. When subfragment 1 formation was inhibited by saturating the Nbs2 [5,5'-dithiobis(2-nitrobenzoic acid)] light chains with Mg2+ ions, chymotrypsin attacked exclusively the HMM junction. The rate of this attack was assessed by measuring the rate of HMM formation by quantitative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by following absorbance changes associated with the solubilization of myofibrillar suspensions. Under rigor conditions, the myofibrils were relatively resistant to the chymotryptic attack. The presence of MgAMP-PNP or MgPPi did not affect the rate of proteolytic attack. On the other hand, binding of MgADP had a powerful stimulating influence on the HMM site digestibility. The dissociation constant for the effect of MgADP was 10 µM < Kd < 50 µM. MgADP did not exercise its unique effect through destabilization of myosin filaments or through dissociation of the actomyosin complex. These results are explained in terms of a change in the myosin cross-bridge conformation brought about by the binding of MgADP to the active site.