A method is presented for the in vivo study of red cell flow dynamics. The method permits direct measurement of the red cell volume fraction in microvessel blood without resort to in vitro calibration curves. Furthermore, the method does not require extensive mathematical manipulation and can be applied to any microvascular network in any tissue. The method also enables direct measurement of red cell velocity, flux, and capillary transit time. Fluorescently labeled erythrocytes in tracer quantities, but known concentrations, are used as indicators of the behavior of the total cell population. Erythrocyte transit time across vascular networks and erythrocyte velocity are determined directly by following the behavior of the labeled cells. Hematocrit and red cell flux are measured by standard microcirculatory methods using labeled cells instead of the total cell population. Data are then converted to absolute values from the measured fraction of labeled cells. The method is thus absolutely dependent on the labeled cells being rheologically normal, and the conditions under which this requirement is satisfied are defined. Microvascular data obtained by the use of this method are presented for hamster cheek pouch and cremaster muscle.