Agonist-induced activation of smooth muscle involves a rise in intracellular Ca²⁺ concentration and sensitization of myosin light chain phosphorylation to Ca²⁺. Sr²⁺ can enter through Ca²⁺ channels, be sequestered and released from sarcoplasmic reticulum, and replace Ca²⁺ in activation of myosin light chain phosphorylation. Sr²⁺ cannot replace Ca²⁺ in facilitation of agonist-activated Ca²⁺-dependent nonselective cation channels. It is not known whether Sr²⁺ can replace Ca²⁺ in small G protein-mediated sensitization of phosphorylation. To explore mechanisms involved in -receptor-activated contractions in smooth muscle, effects of replacing Ca²⁺ with Sr²⁺ were examined in rat portal vein. Norepinephrine (NE) at >3.0 × 10⁷ M in the presence of Ca²⁺ resulted in a strong sustained contraction, whereas this sustained component was absent in the presence of Sr²⁺; only the amplitude of phasic contractions increased. Pretreatment with low (~0.05 mM) free Ca²⁺ followed by 2.5 mM Sr²⁺ resulted in a sustained component of the NE response. In -escin-permeabilized preparations, phenylephrine in the presence of GTP or guanosine 5'-O-(3-thiotriphosphate) alone induced sensitization to Sr²⁺. In conclusion, a Ca²⁺-regulated membrane/channel process is required for the sustained component of NE responses in rat portal vein. Sensitization alone is not responsible for the sustained phase of the NE contraction.