The fluorescent nucleotide analogue of ADP, 1,N6-ethenoadenosine diphosphate (epsilon ADP), has been used to probe the active site of myosin subfragment 1 (SF1). The Mg complex of ADP was shown to be trapped stoichiometrically at the active site by a variety of thiol cross-linking agents having sulfur to sulfur spanning lengths of 2-14 A. Previous studies [Wells, J. A., & Yount, R. G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4966-4970] had suggested ADP was trapped by direct closure of a postulated active site cleft by cross-linking two activity critical thiols, SH1 and SH2. This model was tested by measuring the polarization of trapped and reversibly bound epsilon ADP, the off-rate of trapped epsilon ADP, and the solute quencher accessibility of trapped epsilon ADP on SF1 modified with thiol cross-linking agents of different spanning lengths. The lack of correlation of all of these properties with the length of the cross-linking span suggests that trapping occurs by indirect stabilization of a conformation favoring bound nucleotides rather than by sterically preventing the release of nucleotide. Measurement of the fluorescent properties of epsilon ADP bound to SF1 vs. epsilon ADP free gave a 20% increase in emission intensity, a 7-nm blue shift in the emission maximum, and a 70% increase in the absorbance at the excitation wavelength (330 nm). Trapping of epsilon ADP by the thiol cross-linking agent p-phenylenedimaleimide gave a further 24% increase in emission intensity. This change was shown to be the result of an increase in absorbance of trapped epsilon ADP at 330 nm rather than an increase in the quantum yield.(ABSTRACT TRUNCATED AT 250 WORDS)