Measurements of the intracellular diffusion coefficients (Di) of ATP and creatine phosphate (PCr) in stable, isolated preparations of skeletal muscle were made by means of pulsed field gradient (PFG) 31P NMR. Experiments used a PFG NMR probe specifically designed for small, superfused biological samples. This provided a magnetic field gradient in the z axis of up to 195 G/cm with minimal eddy currents. DiATP and DiPCr in white (fast, glycolytic) skeletal muscle from goldfish (Carassius auratus) were determined to be 2.48 +/- 0.33 and 3.49 +/- 0.33 x 10(-6) cm2/s, respectively, at 25 degrees C and a diffusion time of approximately 19 ms. For comparison with Di values, diffusion coefficients of ATP and PCr also were measured in solutions of ionic composition similar to that of fish muscle cytosol. The in vitro diffusion coefficients of ATP and PCr were 3.54 +/- 0.11 and 5.28 +/- 0.08 x 10(-6) cm2/s, respectively, at 25 degrees C.