The diffusion of beta-enolase and creatine phosphokinase in muscle cells has been studied by modulated fringe pattern photobleaching. Beta-enolase is mobile in the sarcoplasm. At 20 degrees C, the diffusion coefficient is 13.5 +/- 2.5 microm2 s(-1) in the cytosol and 56 microm2 s(-1) in aqueous media. As in the case of dextrans of the same hydrodynamic radius, its mobility is hindered by both the crowding of the fluid phase of the cytoplasm and the screening effect due to myofilaments. A fraction of creatine phosphokinase is mobile in the sarcoplasm. Its diffusion coefficient in the cytosol, 4.5 +/- 1 microm2 s(-1), is lower than that of the dextran of equivalent size. The other fraction (20 to 50%) is very slightly mobile, with an apparent diffusion coefficient varying from 0.0035 to 0.043 microm2 s(-1). This low mobility might be attributed to exchange between free and bound creatine phosphokinase. The bound fraction of the endogenous enzyme was localized by immunocytofluorescence on the cultured muscle cells. Our results favor a localization of bound cytosolic creatine phosphokinase on the M-line and a diffuse distribution in all myotubes.