The hydrogen ion is an important factor in the alteration of vascular tone in pulmonary circulation. Endothelial cells modulate vascular tone by producing vasoactive substances such as prostacyclin (PGI₂) through a process depending on intracellular Ca²⁺ concentration ([Ca²⁺]_i). We studied the influence of CO₂-related pH changes on [Ca²⁺]_i and PGI₂ production in human pulmonary artery endothelial cells (HPAECs). Hypercapnic acidosis appreciably increased [Ca²⁺]_i from 112 ± 24 to 157 ± 38 nmol/l. Intracellular acidification at a normal extracellular pH increased [Ca²⁺]_i comparable to that observed during hypercapnic acidosis. The hypercapnia-induced increase in [Ca²⁺]_i was unchanged by the removal of Ca²⁺ from the extracellular medium or by the depletion of thapsigargin-sensitive intracellular Ca²⁺ stores. Hypercapnic acidosis may thus release Ca²⁺ from pH-sensitive but thapsigargin-insensitive intracellular Ca²⁺ stores. Hypocapnic alkalosis caused a fivefold increase in [Ca²⁺]_i compared with hypercapnic acidosis. Intracellular alkalinization at a normal extracellular pH did not affect [Ca²⁺]_i. The hypocapnia-evoked increase in [Ca²⁺]_i was decreased from 242 ± 56 to 50 ± 32 nmol/l by the removal of extracellular Ca²⁺. The main mechanism affecting the hypocapnia-dependent [Ca²⁺]_i increase was thought to be the augmented influx of extracellular Ca²⁺ mediated by extracellular alkalosis. Hypercapnic acidosis caused little change in PGI₂ production, but hypocapnic alkalosis increased it markedly. In conclusion, both hypercapnic acidosis and hypocapnic alkalosis increase [Ca²⁺]_i in HPAECs, but the mechanisms and pathophysiological significance of these increases may differ qualitatively.