We have performed electron paramagnetic resonance (e.p.r.) experiments on spin-labelled myosin heads in glycerinated insect flight muscle fibres and myofibrils in rigor. Conventional e.p.r. was used to determine the orientation distribution of spin labels relative to the fibre axis, and saturation transfer e.p.r. was used to determine the submillisecond rotational mobility. An iodoacetamide spin label has previously been shown to react selectively with a reactive SH group on the myosin heads of rabbit skeletal fibres, and this label appeared to react with a similar group in insect fibres. Although selective labelling of this group was achieved in insect fibres and myofibrils, the reaction proceeded more slowly than in skeletal muscle, making it more difficult to label myosin heads selectively in insect muscle. The fraction of spin labels bound to myosin was 0.88±0.07 in insect fibres and 0.97±0.05 in rabbit. The fraction of myosin heads labelled was 0.65±0.15 for insect and 0.81±0.10 for rabbit. Both conventional and saturation transfer e.p.r. spectra of insect myosin, myofibrils and fibres were very similar to those of rabbit. The orientation distribution of spin labels relative to the fibre axis in rigor was narrow (16° for rabbit, 22° for insect), and the centre of the angular distribution was essentially the same for insect as for rabbit (68°-69°). This high degree of orientation was accompanied by strong immobilization of the probe on the microsecond time scale. The same immobilization was observed for rigor myofibrils as for purified myosin in the presence of excess actin, but considerable microsecond rotational motion was observed in myosin filaments free of actin. Thus, in insect as well as rabbit, assuming that the labelled heads are representative of all heads, more than 80% of the myosin heads appear to bind to actin in rigor, and the actomyosin bonds are rotationally rigid and oriented within a narrow angular range with respect to the fibre axis.