Previous studies from our laboratory have shown that the average arteriolar pO2 in the hamster skinfold preparation is lower than arterial systemic pO2. In the present work we tested the hypothesis that there is a longitudinal gradient of pO2 along precapillary vessels. Experiments were performed in Syrian golden hamsters bearing a dorsal skin chamber. The oxygen-dependent quenching of phosphorescence of palladium-porphyrin complexes was used to measure intravascular pO2 in the microcirculation. Arterioles were classified in four orders according to their position in the network, first-order vessels being the entrance points. Simultaneous determinations of diameter (D), red blood cell velocity, and systemic blood gases were also made. There was a significant fall of pO2 between vessels of different orders. First-order arterioles (mean D = 64 microns) had pO2 of 51.8 +/- 9.8 mm Hg (mean +/- SD) which was equivalent to approximately equal to 73% of the arterial systemic pO2. Within the arteriolar network, further decreases of intravascular pO2 were measured, leading to a pO2 of 34.0 +/- 7.9 mm Hg in terminal arterioles (mean D = 7.7 microns). In some vessels pO2 was measured in different positions of the same arteriole. The average longitudinal arteriolar oxygen saturation gradient was 3.4 +/- 0.4 delta %/mm (range 0.8-7.2). A significant and positive correlation was found between pO2 and microhemodynamic parameters when arterioles were grouped according to their order. This relation was not significant for venules which showed a mean pO2 of 30.8 +/- 10.8 mm Hg. Tissue pO2 averaged 24.6 +/- 5.8 mm Hg. We conclude that: (1) There is an oxygen loss in arterial vessels larger than 100 micrograms in diameter, (2) arteriolar pO2 in this preparation depends on the position of the vessel within the network, (3) a substantial portion of oxygen delivery to the hamster skin is provided by the arteriolar network, and (4) only a small pO2 gradient exists between terminal arterioles and venules, suggesting that the contribution of the capillary network to tissue oxygenation is relatively small.