Rabbit thoracic aorta was used to determine the effects of decreasing PO<sub>2</sub> on the mechanical properties of contractions in response to norepinephrine (NE) and KC1. Aortae were aerated with 45% O<sub>2</sub>/5% CO<sub>2</sub>/50% N<sub>2</sub> and stimulated with 10 µ M NE or 50 m M KCl. At 5 min of stimulation, 5% CO<sub>2</sub>/95% N<sub>2 </sub>aeration was introduced for 15 min, defined as hypoxia. This time period was previously shown to produce similar decrease in [ATP] in either stimulation condition. Force, stiffness and isotonic shortening velocity were monitored during the initial stimulation, during hypoxia and during re-oxygenation. Hypoxia produced a substantial and rapid decrease in force and a concomitant decrease in stiffness during NE stimulation; delayed and smaller decreases in force and stiffness were observed during KC1 stimulation. The force-stiffness relationship was steeper during KC1 than NE stimulation, and hypoxia did not affect these relationships. Isotonic shortening velocity was significantly depressed by hypoxia during both stimulations although the decrease during KC1 stimulation required a longer time. These data demonstrate that relaxation of an agonist-induced contraction in response to hypoxia results from a decrease in the number of activated crossbridges and not formation of rigor bridges.