Videomicroscopic methods with off-line analysis of microcirculatory parameters by multifunctional computer-assisted image analysis systems have significant advantages for in vivo microvascular research. A limitation of these methods is, however, that red blood cell velocities (V<sub>RBC</sub>) exceeding 2 mm/s cannot be measured using standard video framing rates. In the present study, a high-speed video camera, recording up to 600 frames per second, was incorporated in the set-up, and V<sub>RBC</sub> was measured off-line with the line-shift-diagram method. The aim of this study was to test the reproducibility and validity of the method using a high-speed video camera and to evaluate its applicability in vivo. V<sub>RBC</sub> were measured in arterioles of the split hydronephrotic kidney. The intra- and interindividual variability was small for V<sub>RBC</sub> below 40 mm/s. The validity of the method was tested using the mass conservation principle and found to be at least as good as that of the dual-slit photometric technique. The present approach extends the application of videomicroscopy coupled to image analysis systems to the analysis of high V<sub>RBC</sub>.