We previously reported the relationship between α₁-adrenoceptor-mediated contraction and phosphorylation of 20-kDa myosin light chain (LC₂₀) in de-endothelialized rat caudal arterial smooth muscle at room temperature (Mita M, Walsh MP. Biochem J. 1997;327:669-674). We now describe the effect of increasing the temperature to 37°C on this relationship. The EC₅₀ value (76.6 ± 18.2 nM) for cirazoline (α₁-adrenergic agonist)-induced contraction of the strips at room temperature (23°C) was significantly greater than that (14.5 ± 1.9 nM) at 37°C. The initial rate of the contraction to a sub-maximal concentration of cirazoline (0.3 μM) was similar at the two temperatures. However, cirazoline-induced maximal force at 37°C was approximately 1.8 times that at room temperature. LC₂₀ phosphorylation in response to cirazoline at room temperature and 37°C closely matched the time courses of contraction, but values were not significantly different at the two temperatures: resting phosphorylation levels were 0.09 ± 0.04 mol P_i/mol LC₂₀ at 37°C and 0.22 ± 0.06 mol P_i/mol LC₂₀ at room temperature; maximal cirazoline-stimulated LC₂₀ phosphorylation levels were 0.58 ± 0.08 mol P_i/mol LC₂₀ at room temperature and 0.49 ± 0.05 mol P_i/mol LC₂₀ at 37°C. We conclude, therefore, that the enhanced cirazoline-induced contraction at 37°C is not due to increased LC₂₀ phosphorylation.