We have used electron paramagnetic resonance (EPR) to determine the effects of ADP on the orientational distribution of nitroxide spin labels attached to myosin heads in skinned rabbit psoas muscle fibers. To maximize the specificity of labeling, we spin-labeled ted myosin heads (subfragment 1) on a single reactive thiol (SH1) and diffused them into unlabeled muscle fibers. To maximize spectral and orientational resolution, we used perdeuterated spin labels, 2H-MSL and 2H-IASL, eliminating superhyperfine broadening and thus narrowing the line widths. Two different spin labels were used, with different orientation relative to the myosin head, to ensure that the results are not affected by unfavorable probe orientation. In rigor, a very narrow three-line spectrum was observed for both spin labels, indicating a narrow orientational distribution, as reported previously (Thomas and Cooke, 1980). ADP induced very slight changes in the spectrum, corresponding to very slight (but significant) changes in the orientational distribution. These changes were quantified by a digital analysis of the spectra, using a two-step simplex fitting procedure (Fajer et al., 1990). First, the magnetic tensor values and line widths were determined by fitting the spectrum of a randomly oriented sample. Then the spectrum of oriented fibers was fit to a model by assuming a Gaussian distribution of the tilt angle (θ) and twist angle (∅) of the nitroxide principal axes relative to the fiber axis. A single-Gaussian distribution resulted in inadequate fits, but a two-component model gave excellent results. ADP induces a small (< 5°) rotation of the major components for both spin labels, along with a similarly small increase of disorder about the average positions.