Flow resistance in vivo in small vessels was found unexpectedly high compared to glass tubes of comparable diameters. The main reason for this discrepancy is the presence of a thick endothelial surface layer (ESL). In the present study, we try to derive the effective ESL-thickness from observed flow distributions within microvascular networks. Three microvascular networks in the rat mesentery were used with 392, 546 and 383 vessel segments, for which vessel diameters, network architecture, flow velocity and hematocrit had been determined by intravital microscopy. A mathematical hemodynamic simulation was used to predict flow and hematocrit distributions for different assumptions on ESL thickness. These assumptions were optimized by minimizing deviations of predicted values for velocities, flow directions and hematocrits from measured data. Results indicate that the layer thickness increases from 0 to about 0.8 mm for vessel diameters increasing from 2.4 mm to 10 mm and up to 1 mm for vessel diameters of about 40 mm. An additional hematocrit-dependent impact on flow resistance exhibiting a maximum for vessel diameters around 10 mm is suggested. Thus, the ESL thickness varies with vessel diameter and plays an important role in determining flow resistance in vivo.