A new experimental approach was developed to obtain the first direct measurements of the axial countercurrent thermal equilibration in a microvascular tissue preparation using high resolution infrared thermography. Detailed surface temperature measurements were obtained for an exteriorized rat cremaster muscle in which pharmacological vasoactive agents were used to change the local blood flow Peclet number from 1 to 14 in the feeding artery. Under normal conditions, only the 1A arteries (> 70 microns diameter) showed thermal nonequilibration with the surrounding tissue. The theoretical model developed by Zhu and Weinbaum (28) for a two-dimensional tissue preparation with arbitrarily embedded countercurrent vessels was modified to include axial conduction and the presence of the supporting glass slide. This modified model was used to interpret the experimental results and to relate the surface temperature profiles to the bulk temperature profiles in the countercurrent artery and vein and the local average tissue temperature in the cross-sectional plane. Surface temperature profiles transverse to the vessel axis are shown to depend significantly on the tissue inlet temperature. The eigenfunction for the axial thermal equilibration depends primarily on the blood flow Peclet number and the environmental convective coefficient. The theoretical results predict that when rho(ar)*Pe is less than 1 mm (the range in our experiments), axial conduction is the dominant mode of axial thermal equilibration. For 1 < rho(ar)*PE < 3 mm, countercurrent blood flow becomes comparable to axial conduction, whereas, when rho(ar)*Pe > 3 mm, countercurrent blood flow is the dominant mode of axial thermal equilibration. Therefore, for rho(ar)*Pe > 3 mm the axial equilibration length is proportional to the blood flow Peclet number, as predicted previously by Zhu and Weinbaum in a study in which axial conduction was neglected. It also is shown that the axial decay of the tissue temperature at low perfusion rates can be described by a simple one-dimensional Weinbaum-Jiji equation with a newly derived conduction shape factor.