The interaction of rose bengal (RB) with rabbit skeletal muscle phosphorylase b (1,4-alpha-D glucan: orthophosphate alpha-glucosyl-transferase, E.C. 2.4.1.1.) was studied by kinetic and absorption photometric methods. RB inhibited the phosphorylase b activity. Inhibition was strictly competitive with respect to substrate G-1-P and activator AMP with inhibition constants 2 x 10(-6) M and 2.2. x 10(-7) M, respectively. The association of the dye with the enzyme elicited a red shift in the spectrum of RB indicating an apolar binding site. According to difference absorption measurements, the enzyme binds two dye molecules per dimer in the presence and absence of both G-1-P and AMP. Binding constants determined from photometric titrations are consistent with those obtained from kinetic measurements. The present findings allow to carry out detailed kinetic investigations on the activator AMP and substrate G-1-P binding of phosphorylase b.