Hypotonic swelling increases the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in vascular smooth muscle cells (VSMC). The source of this Ca²⁺ is not clear. To study the source of increase in [Ca²⁺]_i in response to hypotonic swelling, we measured [Ca²⁺]_i in fura 2-loaded cultured VSMC (A7r5 cells). Hypotonic swelling produced a 40.7-nM increase in [Ca²⁺]_i that was not inhibited by EGTA but was inhibited by 1 µM thapsigargin. Prior depletion of inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ stores with vasopressin did not inhibit the increase in [Ca²⁺]_i in response to hypotonic swelling. Exposure of ⁴⁵Ca²⁺-loaded intracellular stores to hypotonic swelling in permeabilized VSMC produced an increase in ⁴⁵Ca²⁺ efflux, which was inhibited by 1 µM thapsigargin but not by 50 µg/ml heparin, 50 µM ruthenium red, or 25 µM thio-NADP. Thus hypotonic swelling of VSMC causes a release of Ca²⁺ from the intracellular stores from a novel site distinct from the IP₃-, ryanodine-, and nicotinic acid adenine dinucleotide phosphate-sensitive stores.