We have studied the binding of ADP and adenosine 5'-[/?,y-imido]triphosphate (AdoPP[NH]P) to insect flight muscle fibrils. We find that 25% of the myosin heads, presumably those which do not interact with actin, bind AdoPP[NH]P with a binding constant greater than 3 x lo6 M-', similar to the binding constant of the same compound to the rabbit myosin heads which do not overlap with actin. The remaining heads in insect myofibrils bind AdoPP[NH]P with an association constant of 8 x lo3 M-', which is eight times stronger than the affinity of this compound for rabbit myosin heads in overlap with actin. Therefore, in contrast to the situation with rabbit myofibrils where AdoPP[NH]P binds much more weakly than ADP, with insect myofibrils these two adenosine phosphates bind with almost equal affinity. This is consistent with the numerous structural studies on insect flight muscle which were interpreted on the basis that most of the actomyosin sites were saturated with nucleotide at an AdoPP[NH]P concentration of 1 mM.The highly regular structure of insect fibrillar flight muscle has made it useful for X-ray diffraction and electron microscopy studies of myosin crossbridge conformation in the presence and absence of nucleotides [l, 21. The most extensively studied condition occurs in the absence of ATP [3 -51 ; this rigor state is widely used as a model for the end of the crossbridge power stroke (the 45" state). The beginning of the power stroke has been modeled after the 90" detached crossbridges seen in Lethocerus muscle relaxed by ATP [3, 61. Attempts to visualize intermediate crossbridge conformations have centered around the use of nucleotide analogs to trap myosin heads at some position between the 90" and 45" states. Adenosine 5'-[P,y-imido]triphosphate (AdoPP[NH]P), an ATP analog not hydrolyzed by myosin [7], is the most widely used of these analogs [8,9] because it causes a large drop in rigor tension without reducing stiffness. This suggests that a large number of crossbridges remain attached but are caught at a different part of the crossbridge stroke. In Lethocerus flight muscle the structure resulting from exposure to AdoPP[NH]P at 4 "C defies simple interpretation because, in the cold, AdoPP[NH]P induces a mixed structural state in which attached crossbridges are altered by the analogue and show both rigor and relaxed features. In addition, although muscles from other species show a drop in tension with constant stiffness in the presence of AdoPP[NH]P, structural responses are varied [lo] or even undetectable 1111.Moreover, the binding of AdoPP[NH]P shows a strong dependence on whether or not the myosin head is bound to