While arteriolar contraction is dependent on Ca²⁺- induced myosin phosphorylation other mechanisms including Ca²⁺ sensitization and time-dependent phenomena such as cytoskeletal and cellular reorganization may contribute to contractile events. We hypothesized that if arteriolar smooth muscle exhibits time-dependent behavior that this may be manifested in differences in relaxation following short and long-term exposure to contractile agonists. Studies were conducted in isolated arterioles, pressurized to 70 mmHg. In initial experiments (n = 10), rate of relaxation was measured following acute (5 min) or prolonged (4 hr) exposure to 5µM norepinephrine (NE). Prolonged exposure to NE resulted in a significantly (P < 0.05) increased time for relaxation in physiological salt solution. Rapid relaxation of vessels exposed to NE for 4 hrs was observed following superfusion with 0 mM Ca²⁺ buffer indicating that the alteration in relaxation was reversible and Ca²⁺ dependent. A similarly impaired dilation was not observed with 4 hr exposure to KCl (75 mM). To determine mechanisms contributing to the effects of prolonged NE exposure studies were performed in the presence of the microtubule depolymerizing agent demecolcine (10µM) or a series of tyrosine phosphorylation inhibitors. While demecolcine caused significant vasoconstriction (P < 0.05) and potentiated NE vasoconstriction it did not prevent the effect of long-term NE exposure on relaxation. Genistein, while having no effect on acute NE-induced contraction, concentration-dependently inhibited prolonged NE constriction. Similarly, the Src (PP1) and p42/44 MAP kinase (PD98059) inhibitors prevented maintenance of long-term NE contraction. The data indicate that prolonged exposure to NE induces biochemical alterations that impair relaxation following removal of the agonist. The contractile effects are Ca²⁺ dependent, involve tyrosine phosphorylation, but do not appear to involve the polymerization state of the microtubule network.