Using vascular smooth muscle, we describe the actions of three pharmacological tools, cyclopiazonic acid (CPA), thapsigargin (TG) and 2,5-di-( tert-butyl)-1,4-benzohydroquinone (tBHQ), which are presumed to act as selective inhibitors of the sarco-endoplasmic reticulum Ca 2+-ATPases (SERCAs). In porcine aortic smooth muscle microsomes two Ca 2+-ATPase activities have been described, one vanadate-sensitive and one vanadate-resistant, representing the Ca 2+-ATPase activities of the plasma membrane and SERCAs, respectively. In agreement, CPA, TG and tBHQ, in the concentration range 0.1 μM to 0.1 mM, dose-dependently inhibit the Ca 2+-ATPase activity only in the vanadate-resistant microsomes. However, 0.1 mM tBHQ also significantly inhibited the Ca 2+-ATPase activity of vanadate-sensitive microsomes. In rabbit aortic rings, all three SERCA inhibitors produced a dose-dependant inhibition of contractions evoked by 20 mM caffeine or 1 μM phenylephrine (PE) in a Ca 2+-free physiological solution. However, in PE-contracted rings, tBHQ (≥30 μM) also significantly inhibited the ability of cromakalim to induce relaxation. In conclusion, the data suggest that CPA, TG and tBHQ can all act as selective SERCA inhibitors in both porcine and rabbit aortic smooth muscle. However, in contrast to CPA and TG, high concentrations of tBHQ can exhibit some nonspecific effects, which include inhibition of the plasma membrane Ca 2+-ATPase and possibly K + channels regulated by cromakalim.