The ability of extracellular Ca2+ to refill internal Ca2+ stores of canine tracheal smooth muscle after a prior depletion by acetylcholine (ACh) was assessed using a novel sarcoplasmic reticulum (SR) Ca2+ pump inhibitor, cyclopiazonic acid (CPA). The transient contraction induced by ACh in a medium free of Ca2+ was used as an index for the content of agonist-sensitive intracellular Ca2+ stores. CPA inhibited in a concentration-dependent manner the refilling of the stores occurring during high KCl stimulation, and this inhibitory effect was independent of the external Ca2+ concentration. On the other hand, CPA was less effective in inhibiting the refilling occurring during prolonged ACh stimulation, especially when external Ca2+ concentration was raised. At 5.0 mM external Ca2+ or when 0.1 microM BAY 8644 was present in the medium, CPA was ineffective in inhibiting the refilling occurring during prolonged ACh stimulation. The maximum ACh-induced contraction in Ca(2+)-containing medium was independent of the extent of internal store Ca2+ load in the absence of L-type Ca2+ channel blocker but was highly dependent on the extent of internal Ca2+ load in the presence of the Ca2+ channel blocker. Hyperpolarization of the plasma membrane with the K+ channel opener cromakalim reduced the amplitude of ACh tonic contraction. Subsequent addition of nifedipine further reduced ACh tonic contraction. It is concluded that two different pathways for external Ca2+ are used to refill ACh-sensitive internal stores. One involves active Ca2+ uptake via a CPA-sensitive Ca2+ pump, and the other involves a CPA-insensitive pathway whose nature remains to be determined.