The binding of [3H]ADP and [3H]adenyl-5'-yl-imidodiphosphate ([3H]AMP-PNP) to rabbit skeletal myofibrils was measured at 25 and 7 degrees C, mu = 0.12 M, using [14C]mannitol as a volume marker. We found that ADP bound to myosin heads in overlap with a binding constant of about 10(4) M-1, similar to the value we previously obtained in vitro with acto.S-1. The binding of AMP-PNP to myosin heads was measured both in and out of overlap. The affinity of AMP-PNP to the heads out of overlap was similar to that obtained in vitro with S-1 alone. The binding of AMP-PNP to the myosin heads in overlap was much weaker. We could fit these data with a binding constant of about 1 x 10(3) M-1, assuming a single population of cross-bridges and 1 mol of AMP-PNP bound per mol of myosin head. This value was reduced by a factor of 2 when we corrected for nonspecific binding. It was also possible to fit the data assuming two equal populations of cross-bridges with one of the populations binding AMP-PNP about 5-fold more strongly than the other population. Therefore, for at least half of the cross-bridges in overlap, the binding of AMP-PNP is almost as weak as the value of 3 x 10(2) M-1 we previously measured for the acto.S-1 complex in vitro (Biosca, J. A., Greene, L. E., and Eisenberg, E. (1986) J. Biol. Chem. 261, 9793-9800).