The value of the diffusion coefficient for oxygen in muscle is uncertain. The diffusion coefficient is important because it is a determinant of the extracellular oxygen tension at which the core of muscle fibers becomes anoxic (PO_2crit). Anoxic cores in muscle fibers impair muscular function and may limit adaptation of muscle cells to increased load and/or activity. We used A.V. Hill's diffusion equations to determine Krogh's diffusion coefficient (D) for oxygen in single skeletal muscle fibers from Xenopus laevis at 20°C (n = 6) and in myocardial trabeculae from the rat at 37°C (n=9). The trabeculae were dissected from the right ventricular myocardium of control (n=4) and monocrotaline-treated, pulmonary hypertensive, rats (n=5). The cross-sectional area of the preparations, the maximum rate of oxygen consumption (VO_2max), and the minimum oxygen tension required to reach VO_2max (PO_2crit) were determined. Krogh's diffusion coefficient increased in the order: Xenopus muscle fibers D=1.23 nM mm²/mmHg s (SD 0.12), control rat trabeculae D=2.29 nM mm²/mmHg s (SD 0.24, P=0.0012 vs. Xenopus) and hypertrophied rat trabeculae D=6.0 nM mm²/mmHg s (SD 2.8, P=0.039 vs. control rat trabeculae). D increased with extracellular space in the preparation (Spearman's rank correlation coefficient = 0.92, P<0.001). The values for D indicate that Xenopus muscle fibers cannot reach VO_2max in vivo because PO_2crit can be higher than arterial PO₂, and that hypertrophied rat cardiomyocytes can become hypoxic at the maximum heart rate.