The unique fast-reacting cysteine residue (SH1) of myosin subfragment 1 (S1), prepared by chymotryptic digestion, and cysteine 373 of actin have been labelled selectively with the fluorescent probes, N-(bromoacetyl)-N'-(1-sulpho-5-naphthyl)ethylenediamine (1,5-BrAEDANS) and 5-(iodoacetamido)fluorescein (5-IAF), whose spectral properties render them a particularly effective donor-acceptor pair in fluorescence energy transfer studies. The transfer efficiency of 40-45% represented a spatial separation of the chromophores of about 5 nm, which is in reasonable agreement with the value of 6 nm reported earlier for similarly labelled S1, prepared by papain digestion, and actin [Takashi, R. (1979) Biochemistry, 18, 5164-5169]. This transfer efficiency did not change when the doubly-labelled binary complex was formed: (1) with acto-S1(A1) or acto-S1(A2) at 10-200 mM KCl, pH 7-8 and different buffer conditions; (2) with either S1 isoenzyme and regulated actin (i.e. actin with tropomyosin and troponin) both in the presence and absence of Ca2+ or when the donor and acceptor attachment sites were reversed. Analysis of donor and acceptor polarized fluorescence showed that the chromophores are not randomly orientated (i.e. chi 2 not equal to 2/3), but they do have some motion relative to either protein. From a knowledge of the limiting values for chi 2, the intersite distance for donor and acceptor chromophores was calculated to be in the range 3.9-6.7 nm. Addition of MgATP to the doubly-labelled acto-S1 complex eliminated energy transfer but this was recovered when ATP hydrolysis was completed. By utilizing the known binding constants between S1, actin and either MgADP or MgAdoPP[NH]P (magnesium adenosine 5'-[beta, gamma-imido]triphosphate) [Konrad, M. and Goody, K. (1982) Eur. J. Biochem. 128, 547-555; Greene, L. E. and Eisenberg, E. (1980) J. Biol. Chem. 255, 543-548], the concentrations of all species present at equilibrium were determined. Experimental conditions were chosen to maximise the amount of ternary acto-S1-nucleotide complex (approximately equal to 50%) and minimise the amount of binary complex (less than or equal to 2%). The spatial separation of the chromophore interaction sites in the ternary complex was found to be the same with both nucleotides and indistinguishable from that found with the binary complex. A similar strategy was employed to compare the conformations of the binary and ternary complexes by 1H-NMR spectroscopy. In these experiments about 90% of the S1 was in the form of the ternary complex. There was no noticeable change in the acto-S1 spectra upon addition of either MgAdoPP[NH]P or MgADP. These observations support the conclusion that there is no large change in structure in the 'rigor' binary acto-S1 complex when it binds either ADP or AdoPP[NH]P.