In autoperfused preparations of feline jejunum, blood flow was measured from the mucosal surface with laser-Doppler velocimetry (LDV) and hydrogen gas (H2) clearance techniques while blood flow was altered by intra-arterial infusions of isoproterenol. LDV and H2 clearance estimates of blood flow were compared with total-wall and mucosal-submucosal blood flows measured with the radiolabeled microsphere technique. Over the range (26.3–73.6 ml X min-1 X 100 g-1) of blood flows attained, a series of direct linear relationships were obtained among LDV, H2 clearance, and microsphere estimates of jejunal blood flow. The slopes of these relationships indicated that the H2 clearance technique over-estimates total intestinal blood flow but reflects mucosal-submucosal flow as measured with microspheres. LDV measurements of blood flow from the mucosal surface were equally well correlated with total and mucosal-submucosal blood flow measured by microspheres, thereby not allowing for a definitive conclusion on the measurement depth of the LDV method. However, the ability of the LDV method to detect changes in blood flow in the perfused gut, even through 3 mm of unperfused tissue, casts a doubt on the assumption that the LDV method has a spatial resolution of less than 0.5–1.0 mm. The results of this study indicate that the H2 clearance technique can be used to measure mucosal blood flow in the small intestine. By contrast, the precise measurement depth of the LDV method is still uncertain and requires further evaluation.