Abstract The polarized fluorescence from nucleotides bound to myosin heads in glycerinated muscle fibers of rabbit psoas was measured as the number of myosin heads with bound nucleotides was varied by adding various concentrations of fluorescent ϵ-ATP, ϵ-ADP and ϵ-AMPPNP (1:N6-etheno-ATP, -ADP and -imido ATP). The angles of the absorption and emission dipoles of bound nucleotides to the fiber axis and their angular distribution were determined from the observed values of four components of the polarized fluorescence. The maximum amount of nucleotides bound to the myosin heads in the fiber, Bm, was 170 to 270 μ m . The dissociation constant of nucleotides, K 1 2 , increased in the order ϵ-ATP, ϵ-ADP, ϵ-AMPPNP, and was four to six times larger at a sarcomere length (SL) of 2.1 μm than at 3.7 μm. The polarized fluorescence from bound ϵ-ADP at SL = 2.1 μm was independent of the amount of bound ϵ-ADP when it was lower than one-half of Bm, indicating a single helical array of myosin heads having ϵ-ADP. The angles of the absorption dipole, φA, and the emission dipole, φE, to the fiber axis were 69 ° and 66 °, respectively. As the amount of bound ϵ-ADP exceeded one-half of Bm, the values of the polarized fluorescence showed that the extra ϵ-ADP bound to myosin heads with a lower affinity and had different angles to the fiber axis: φA and φE were 49 ° and 54 °, respectively. The half-maximum width of the angular distribution of these bound ϵ-ADP molecules, θ 1 2 , was about 20 °. During development of isometric tension in the presence of ϵ-ATP with Mg2+, the polarized fluorescence was independent of the amount of bound ϵ-ATP when it was lower than one-third of Bm or when the concentration of free ϵ-ATP was lower than 100 μ m , indicating a single helical array of myosin heads undergoing the ATPase reaction. The angles of bound nucleotides, φA and φE, were 68 ° and 64 °, respectively. The half-maximum width of the angular distribution, θ 1 2 , was about 22 °. As the amount of bound nucleotides exceeded one-third of Bm, the polarized fluorescence showed deviation from the values expected for the single helical array. The angles φA and φE for bound ϵ-AMPPNP were about 58 ° and 62 °, respectively, but the angular distribution was broad; that is, θ 1 2 was about 42 °. These angles were independent of the amount of bound ϵ-AMPPNP. In a stretched fiber with SL = 3.7 μm, the polarized fluorescence showed that the angles of ϵ-ADP, ϵ-ATP and ϵ-AMPPNP bound to myosin heads had almost random distributions; θ 1 2 was 90 ° to 100 °, independent of the amount of bound nucleotides. Similar results were obtained with the relaxed fiber in the presence of ϵ-ATP.