The respective effects of hyperosmolarity caused by impermeant solutes, such as mannitol and sucrose, on the endothelium and smooth muscles cell responses were investigated in the rat tail artery. The vessels, with or without endothelium, were infused and superfused with an isosmolar saline solution, and were repeatedly stimulated with phenylephrine. Superfusing with hyperosmolar fluid (390–420 mosm/l) produced a transient increase in the arterial basal perfusion pressure which peaked after approximately 5 min and then declined within 15 min to a stable nonsignificant value above control values in subsequent experiments. In arteries with functional endothelium, the effect of phenylephrine was about 1.9-fold larger in hyperosmotic medium compared to that in isosmotic medium. In hyperosmotic media the response was still more than twofold enhanced in endothelium-denuded vessels compared to those with endothelium. In the latter, indomethacin (10 µ M) had no effect, but N<sup>ω</sup>-nitro- L-arginine methylester ( L-NAME; 30 µmol/l), an inhibitor of NO production, enhanced the response to phenylephrine to reach the same magnitude of response as seen in endothelium-denuded arteries. This effect of L-NAME was antagonized by L-arginine. Relaxation induced by the NO donor SIN-1 was unchanged by hyperosmolarity, indicating that the effect of NO was not impaired. It is concluded that, in the rat tail artery, the enhancement in phenylephrine-induced contractions produced in a hyperosmolar solution is due to both an endothelium-independent increase in smooth muscle responses and a moderate decrease in the production of NO, or an NO-like factor, by the endothelium. In spite of this reduction, endothelium-derived NO still plays a major role in attenuating phenylephrine-induced contractions in hyperosmolar medium.