PGE(2) is a powerful modulator of uterine contractility, but there is uncertainty as to which receptor subtypes (EP1, EP2, Er3, or EP4). G proteins, and second messenger systems are activated by PGE(2) in myometrium. Here we show that in cultured human myometrial cells, PGE(2) (1-100 mu m) activates phospholipase C (PLC) up to 500% over the control level and elevates intracellular calcium ([Ca2+](i) from the resting level of 60-90 nM up to 350 nM in a concentration-dependent manner. Stimulation by the receptor. subtype-selective analogs GRG3799X (EP3), sulprostone (EP3 > EP1), and misoprostol (EP3 > EP2 > EP1) indicates that these effects are transmitted through EP3 receptors. Both effects are resistant to pertussis toxin (PT). Lower concentrations of PGE(2) (1-300 nM) increase [Ca2+](i) via a PT-sensitive pathway, without PLC activation. This [Ca2+](i) increase occurs after an inverse dose-related delay and is inhibited by the selective EP1 antagonist AH6809 anti calcium channel blockers. By comparison oxytocin stimulates PLC up to 1000% over the control level and elevates [Ca2+](i) up to 800 nM in a concentration-dependent manner without any measurable delay; both effects are partly sensitive to PT. These data provide functional evidence for the presence of different stimulatory mechanisms for PGE(2) in myometrium: 1) a low affinity receptor (probably EP3D) that activates PLC through a PT-insensitive pathway; and 2) a high affinity receptor (probably EP1), independent from PLC and involving a PT-sensitive G protein (G(i)?). Both pathways lead to elevation of [Ca2+](i).