Orthogonal polarization spectral (OPS) imaging was validated against intravital fluorescence microscopy (IFM) for microvascular measurements in skin flaps of hairless mice. Examinations were performed 1, 6, and 24 hours after elevation (n = 8) with both OPS imaging and IFM. A fluorescent dye was a prerequisite for IFM measurements but not for OPS imaging. Our findings show that OPS imaging can visualize the skin flap microcirculation independent from the application of fluorescent tracers. From these images, quantitative analysis of functional capillary density (FCD) was feasible. As expected, FCD was significantly lower in the distal part of the flap compared with its base (171.8 ± 34.7 versus 62.0 ± 25.6, mean ± SD; 1 hour data). Comparison of OPS imaging and IFM revealed a significant correlation of FCD values (P < 0.001) at all time points. Given the success of this validation study on mouse skin flaps, clinical investigations will have to prove that OPS imaging can also successfully be used to monitor flaps in humans. © 2001 Wiley-Liss, Inc. MICROSURGERY 21:317–324 2001