ABSTRACTObjective:To measure the magnitude of the water‐exclusive pathway in cat skeletal muscle.Methods:The osmotic reflection coefficient (σd) was measured for sucrose, raffinose and cyancobalamine using osmotic transient techniques in the isolated, perfused cat hindlimb preparation at sufficiently high perfusate flows (60–80 ml/min−1100 g−1) so that solute diffusion was not a factor. Microvascular filtration coefficient values required for the σddetermination were measured using the capillary filtration coefficient (CFC) technique at these high flows. With these σddata and macromolecular reflection coefficient data from a previous study, discrete pore‐modeling techniques were used to estimate the magnitude of the water movement through the water‐exclusive pathway.Results:CFC values increased significantly at very high flows (>80 ml/min−1100 g−1), but these values were unchanged from control at the lower flows used to measure σd. The σdvalues for sucrose and raffinose were 0.41 ± 0.03 SE and 0.42 ± 0.03 SE, respectively, in 12 limbs. In the same limbs, the σdfor cyancobalamine was 0.52 ± 0.03 SE, which was significantly (p<0.05) larger, consistent with a larger Stokes‐Einstein radius for this molecule. A 3‐pathway model (small and large pores and a water‐exclusive pathway) was fit to the data. The result was that 41 ± 4% (95% confidence interval) of total water flow makes use of the water‐exclusive pathway in this preparation.Conclusions:The very high fraction of water flow through the water‐exclusive pathway in cat skeletal muscle suggests that this pathway is of major importance in microvascular water movement under normal conditions. Failure to take this finding into account can lead to inaccuracies in the estimation of paramet