The interaction of the hydrophobic probe conjugated polyene fatty acid cis-parinaric acid (PA) with myosin and its fragments was studied by measuring the enhancement of PA fluorescence following binding. The measurements point to the presence of about 1.34 hydrophobic sites per mol of myosin or per mol of double-headed fragment heavy meromyosin and 0.65 site per mol of myosin subfragment 1 (S-1). The binding constants were all in the range of 107 M−1. The S-1 isoenzyme containing alkali light chain 1 [S-1(A1)] bound strongly close to 1 mol of PA per mol of S-1(A1) while the isoenzyme containing alkali light chain 2 [S-1(A2)] bound no PA. Isolated alkali light chains A1 and A2 bound PA very weakly. Mg-nucleotide binding to the S-1 active site had little effect on the stoichiometry or affinity of PA binding; binding of actin to myosin carrying the probe either in solution or in an organized system of myofibrils had no effect on the magnitude of probe fluorescence but dramatically increased the rotational relaxation time of bound PA. These data indicated that parinaric acid bound to the hydrophobic pocket formed between the 41-residue region at the N-terminal end of A1 and the heavy chain of S-1 and that the hydrophobic pocket is distant from both actin and the nucleotide binding sites.