Using Fura PE3-loaded rat intrapulmonary arteries (IPA) from humanely killed rats, we have examined the mechanisms by which prostaglandin F_2α (PGF_2α) increases the intracellular Ca²⁺ concentration [Ca²⁺]_i with simultaneous recording of isometric force. Values are means ± S.E.M. A low concentration (0.1μM) of PGF_2α caused a marked transient rise in [Ca²⁺]_i followed by a sustained plateau, which was not associated with contraction. The plateau was abolished by removal of extracellular Ca²⁺, pre-treatment with La³⁺ (10μM), the putative store-operated channel antagonist 2-APB (75μM), and the SERCA inhibitor thapsigargin (0.1 or 1.0μM), and reduced by 87 ± 5% by the phospholipase C antagonist U-73122 (3μM). It was unaffected by the L-type Ca²⁺ channel blocker diltiazem (10μM) and by the TP receptor antagonist SQ-29548 (1μM). The selective FP receptor agonist fluprostenol (0.1μM) caused an identical response. A higher concentration (10μM) of PGF_2α caused a contraction which was abolished by SQ 29548 and associated rise in [Ca²⁺]_i which was partially inhibited by diltiazem. The diltiazem-resistant component of this increase in [Ca²⁺]_i was reduced by 78 ± 3% and 47 ± 17 % by 75μM 2-APB and 10μM La³⁺, respectively, but by 31 ± 10% by U73122. The rise in [Ca²⁺]_i in response to the TP receptor agonist U-46619 (50nM) was similarly (44 ± 11%) inhibited by 10μM diltiazem, and was inhibited by 43 ± 12% by thapsigargin pre-treatment. When Ca²⁺ was replaced by Sr²⁺ (4mM), only the transient increase in the Fura PE-3 signal occurred following application of 0.1μM PGF_2α. Conversely, both 10μM PGF_2α and 50nM U-46619 caused a sustained increase in Fura PE3 signal similar to that observed in Ca²⁺-containing PSS. These results suggest that at low concentrations PGF_2α acts via FP receptors to cause IP₃-dependent Ca²⁺ release and store operated Ca²⁺ entry (SOCE), resulting in a large rise in [Ca²⁺]_i which is not associated with contraction. U-46619 and higher concentrations of PGF_2α cause a TP receptor-mediated contraction and additional Ca²⁺ influx involving both L-type Ca²⁺ channels and a receptor-operated pathway, which differs from SOCE in that it is less La³⁺ and thapsigargin sensitive, does not require PLC activation, and is Sr²⁺ permeable.