To elucidate the structure of the cross-bridge intermediates in the actomyosin ATPase cycle, several laboratories have added both ethylene glycol and AMP-PNP to muscle fibers. These studies suggested that ethylene glycol shifts the structure of myosin.AMP-PNP toward the weak-binding conformation, i.e., toward the structure of myosin.ATP. Since only the weak-binding conformation of myosin subfragment 1 (S-1) binds with no apparent cooperativity to the troponin-tropomyosin-actin complex (regulated actin), we used this as a probe to examine the conformation of various S-1.nucleotide complexes in ethylene glycol. Our results show that ethylene glycol markedly weakens the binding strength of S-1, S-1.ADP, and S-1.AMP-PNP to actin but has almost no effect on the binding strength of S-1.ATP. As in muscle fibers, at 40% ethylene glycol, the binding strength of S-1.AMP-PNP to actin becomes very similar to the binding strength of S-1.ATP. In the presence of troponin-tropomyosin, the binding of S-1.AMP-PNP to actin shows no apparent cooperativity in 40% ethylene glycol. Therefore, our results confirm that ethylene glycol shifts the structure of the myosin.AMP-PNP toward the weak-binding conformation. However, our results also suggest that ethylene glycol has a direct effect on the regulated actin complex. This is shown by the fact that ethylene glycol markedly increases the cooperative binding of S-1.ADP to regulated actin both in the presence and in the absence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)