In vivo and in vitro studies concerned with the role of oxygen in the regulation of blood flow have primarily investigated the response of blood vessels to decreases in oxygen tension. In this study, we examined the response of isolated rat cremaster skeletal muscle arterioles to increases in oxygen tensions. First-order arterioles with an average diameter of 90 microns were cannulated and pressurized to 65 mm Hg and studied under constant pressure in a no-flow state. Arterioles were equilibrated in a Krebs bicarbonate-buffered solution (ph 7.4) gassed with 21% O2, 5% CO2, 74% N2. Changes in arteriolar diameters were continuously measured and recorded in response to increases in bath PO2 (20 to 660 mm Hg). Arterioles were studied before and after either the removal of the endothelium or the administration of indomethacin (IND, 10(-5) M), to inhibit prostaglandin synthesis. When the bath PO2 was increased from 20 to 150 mm Hg, arteriolar diameters decreased by 37%; they then decreased an additional 14% when bath PO2 was increased from 150 to 660 mm Hg. Removal of the endothelium or administration of IND completely eliminated the arteriolar constrictions in response to increases in PO2 from 20 to 150 mm Hg, and from 150 to 660 mm Hg. These observations suggest that rat cremaster arterioles constrict to increases in oxygen tension by reduction in the synthesis of endothelium-derived dilator prostaglandins.