Cis-parinaric acid (PA) binds to a hydrophobic pocket formed between the heavy chain of myosin subfragment-1 (S1) and the 41-residue N-terminal of essential light chain 1 (A1). The binding is strong (Ka = 5.6 x 10(7) M-1) and rigid (polarization = 0.334). PA does not bind to myofibrils in which A1 has been extracted or replaced with alkali light chain 2 (A2). As in the case of S1 labeled with other probes, polarization of fluorescence of S1-PA added to myofibrils depended on fractional saturation of actin filament with S1, i.e., on whether the filaments were fully or partially saturated with myosin heads. Because fluorescence quantum yield of PA is enhanced manyfold upon binding, and because PA binds weakly to myofibrillar structures other then A1, the dye is a convenient probe of cross-bridge orientation in native muscle fibers. The polarization of a fiber irrigated with PA was equal to the polarization of S1-PA added to fibers at nonsaturating concentration. Cross-linking of S1 added to fibers at nonsaturating concentration showed that each S1 bound to two actin monomers of a thin filament. These results suggest that in rigor rabbit psoas muscle fiber each myosin cross-bridge binds to two actins.