The aims of the present study were to establish whether vasoactive intestinal polypeptide (VIP) could mobilize internally-stored Ca²⁺ and whether Ca²⁺ release could trigger Ca²⁺ influx from the extracellular space. Bovine pulmonary artery endothelial cells from an established cell line were loaded with fura-2/AM and cells were studied in suspension or were imaged in monolayers at 40–80% confluency. In Ca²⁺ imaging studies, VIP evoked Ca²⁺ transients in Ca²⁺-free medium containing 50 μM EGTA. This was observed in 33 out of 122 cells examined on 29 separate trials. With each cell, the spread of Ca²⁺ appeared to occur from the periphery of the cell to the central core. Cells which did not respond to VIP responded to other stimulants such as bradykinin, endoplasmic reticulum Ca²⁺ pump inhibitors, (cyclopiazonic acid and thapsigargin), and endoplasmic reticulum Ca²⁺ release channel opener, ryanodine. The reintroduction of Ca²⁺ following VIP-induced Ca²⁺ release did not evoke a Ca²⁺ response in 5 cells imaged. Cells in suspension showed typical biphasic responses to bradykinin, thapsigargin or cyclopiazonic acid in the presence of external Ca²⁺. Stimulation with VIP caused transient Ca²⁺ responses in Ca²⁺-free physiological saline containing 50 μM EGTA. However, only 1 out of 4 cells tested showed a response to Ca²⁺ when it was reintroduced to the bathing medium. This study provided direct evidence for the first time in these bovine endothelial cells for VIP-mediated elevation of cytosolic concentration of Ca²⁺. The results also suggested that other mechanisms might prevail preventing capacitative Ca²⁺ entry following the release of internally-stored Ca²⁺.