Fresh frozen skeletal muscles of rats, rabbits, and humans were sectioned in a cryostat. Sections 12 to 32 micra thick were incubated in a substrate solution for the histochemical demonstration of non-specific alkaline phosphatase activity. A modified azo dye coupling technique was used at pH 9.5. Localized areas of high enzymatic activity were found in specific and well defined areas along the terminal arterial tree, in addition to the activity which has been previously described in capillary endothelium. Arterial branches with luminal diameters of 25 micra or less showed staining of their endothelium starting abruptly at their origin from the parent vessel and fading distally. Smaller arterial branches showed the same localization of enzymatic activity and stained more intensely. Other organs of rats surveyed showed arterial branches with the same pattern of staining. Identical results were obtained using the Gomori technique for alkaline phosphatase. Extensive saline perfusion of the vascular tree did not affect the observed localization of enzymatic activity. The enzymatic activity described may be part of the mechanism regulating the blood flow.