Proteases in fish muscle often cause undesired softening of intact meat pieces during refrigerated storage or slow cooking. Several food-grade proteinaceous inhibitors can overcome this softening if properly delivered to the intracellular sites where proteases are located. Fluorescence recovery after photobleaching (FRAP) and laser scanning confocal microscopy (LSCM) were used to measure the translational diffusion of fluorescein isothiocyanate (FITC)-labeled protease inhibitors into intact muscle fibers of halibut. Diffusion coefficients (D) of alpha-2-macroglobulin (720 kDa), soybean trypsin inhibitor (21 kDa), and cystatin (12 kDa) were measured in both muscle fibers and dilute aqueous solutions. On the time scale of the observation (35 min), cystatin and soybean trypsin inhibitor diffused through the cell membrane (sarcolemma) and sarcoplasm, but at a considerably slower rate (>10-fold difference) than in dilute aqueous solution. alpha-2-Macroglobulin did not diffuse into muscle cells within the time frame of the experiment, but did completely penetrate the cell during overnight exposure. The present study thus shows a clear dependence of D on protein inhibitor size when moving within intact skeletal muscle fibers. Low molecular weight protease inhibitors such as cystatin can be effectively diffused into intact fish muscle cells to minimize proteolytic activity and meat softening.