A modified preparation of the rat spinotrapezius muscle is described in which optimal conditions for intravital microscopy can be achieved while the supplying blood vessels are left fully intact, and mechanical stress to the muscle during preparation is reduced to an unavoidable minimum. The viability of the preparation is demonstrated using the response of arterial microvessels to endothelium-dependent and -independent dilators and to changes of ambient PO2, the presence of spontaneous vasomotion, and histochemical analysis of pertinent enzyme systems. The preparation is viable for much longer experimental time periods (up to 10 hours) than reported previously, provided the intensity of illumination is kept at a very low level. If the latter prerequisite is met, tissue edema, maximal vasodilation, and the associated loss of responsiveness to vasoactive stimuli of arterioles is reliably avoided.