Ca<sup>2+</sup> inflow via store-operated Ca<sup>2+</sup> channels was investigated in rings of rat tail and basilar arteries kept in serum-free organ culture, which is known to preserve the contractility of the vascular smooth muscle. After culture for 3–4 days, Ca<sup>2+</sup> release from intracellular stores in response to caffeine (20 m M) was augmented 2- to 4-fold. Following depletion of intracellular Ca<sup>2+</sup> stores by caffeine and thapsigargin (10 µ M), addition of Ca<sup>2+</sup> (2.5 m M) caused an increase in the intracellular Ca<sup>2+</sup> concentration which was 2–3 times greater in cultured than in freshly dissected rings, and was not affected by verapamil (10 µ M). In contrast, L-type Ca<sup>2+</sup> channel currents were decreased by 20% after culture. While freshly dissected rings developed no or very little force in response to the addition of Ca<sup>2+</sup> after store depletion, cultured rings developed 42% (tail artery) and 60% (basilar artery) of the force of high-K<sup>+</sup>-induced contractions. These contractions in cultured vessels were insensitive to verapamil but could be completely relaxed by SKF-96365 (30 µ M). Store depletion by caffeine increased the Mn<sup>2+</sup> quench rate 3- to 4-fold in freshly dissected as well as cultured tail artery, while there was no increase in freshly dissected basilar artery, but a 3-fold increase in cultured basilar artery. Uptake of Ca<sup>2+</sup> into intracellular stores was twice as rapid in cultured as in freshly dissected tail artery. This study shows that organ culture of vascular smooth muscle tissue causes changes in Ca<sup>2+</sup> handling, resembling the pattern seen in dedifferentiating smooth muscle cells in culture, although contractile properties are maintained.