Based on change in anisotropy of fluorescently labelled S1 and on increase in turbidity of acto-S1 complex when S1 bound to F-actin, we reported previously that depending on the molar ratio of S1 to actin two different complexes of actin monomer (A) and myosin subfragment 1 (S1) could be formed: A1*S1 (one actin with one S1) and A2*S1 (two actins with one S1). Here we extend these findings to F-actin labelled with pyrene and cross-linked to S1 with 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC). The fluorescence of pyrene F-actin decreased with increase in S1 concentration and reached saturation at a molar ratio of S1 to actin of either 0.5 or 1.0, depending on whether S1 was added slowly (5 min) or quickly (10-20 s between additions). Incubation of A2*S1 complex in excess of S1 for > 1 h caused a shift in equilibrium towards the A1*S1 complex. The A2*S1 complexes were not formed at high S1 to actin ratios (>1.0) owing to competition between heads. Crosslinking experiments showed that the formation of EDC crosslinked products, 175-185 kDa doublet and 265 kDa band, depended on the ratio S1 to actin. To assess the relative ratio of S1 and actin in crosslinked products, we labelled S1 and F-actin with different fluorescent probes (5-IAF and IATR). The S1 to actin ratio was proportional to the ratio of intensities of fluorescence of labelled S1 and actin. The S1 to actin ratio in 265 kDa product was two times smaller than in 175-185 kDa doublet (which is believed to be A1*S1 complex) and therefore 265 kDa band corresponded to A2*S1. Transition between two types of binding may be important to understanding how muscle contracts.