Specific vasoactive substances such as endothelin (ET) have been proposed to induce heterogeneity of tissue perfusion and thus the oxygen delivery at the sinusoidal level in the liver, but a direct method for testing this hypothesis has not been available. Our objective was to develop a method to test the hypothesis that functional heterogeneity of blood flow can be induced at the sinusoidal level by mediators such as endothelin-1, which act at the sinusoidal level. We constructed oxygen-sensitive membranes using tris (1,10-phenanthroline) ruthenium (II) chloral hydrate, a dye whose fluorescence is quenched by oxygen incorporated into a silicon rubber membrane. The membrane (less than 40 microm thick) was formed on a glass coverslip that served as the viewing window of the system for in vivo fluorescence microscopy and allowed determination of the PO2 distribution in rat liver acini during intraportal infusion of ET or phenylephrine (PE) in vivo. Heterogeneity was quantified by comparing the coefficient of variation (CV) of the fluorescence intensity within the zone 1 before, during, and after drug infusion. PE and ET doses were matched to produce a similar increase in portal pressure. PE caused a gradient of PO2 across zones, but within zone 1 no significant increase in CV was observed. In contrast, ET produced a patchy pattern of both an increase and decrease in PO2 resulting in doubling (P < 0.01) in CV of fluorescence intensities within zone 1. These results indicate that PE, which acts at presinusoidal sites, results in a homogeneous decrease in tissue PO2 within a zone, while ET, which additionally acts at sinusoidal sites, induces significant microheterogeneity of tissue PO2. The oxygen-sensitive membrane provides a useful tool for oxygen mapping in vivo.