Prostaglandin E2 (PGE2) imparts diverse physiological effects on multiple airway cells through its actions on four distinct E-type prostanoid (EP) receptors (EP1-4). Gs-coupled EP2 and EP4 receptors are expressed on airway smooth muscle (ASM), yet their capacity to regulate ASM contractile state remains subject to debate. We used EP2 (ONO-259) and EP4 (ONO-329) subtype-specific agonists in cell- and tissue- based models of human ASM contraction (Magnetic Twisting Cytometry (MTC), and precision cut lung slices (PCLS), respectively) to study the EP2- and EP4- regulation of ASM contraction and signaling under conditions of histamine or methacholine (MCh) stimulation. ONO-329 was superior (<0.05) to ONO-259 in relaxing MCh-contracted PCLS (logEC50: 4.9e-07 vs. 2.2e-06; maximal bronchodilation 35± 2% S.E. vs. 15± 2% S.E.). However, ONO-259 and ONO-329 were similarly efficacious in relaxing histamine-contracted PCLS. Similar differential effects were observed in MTC studies. Signaling analyses revealed only modest differences in ONO-329- and ONO-259- induced phosphorylation of the PKA substrates VASP and HSP20 with concomitant stimulation with MCh or histamine. Conversely, ONO-259 failed to inhibit MCh- induced pMLC20 and the F/G actin ratio, while effectively inhibiting their induction by histamine. ONO-329 was effective in reversing induced pMLC20 and F/G actin by both MCh and histamine. Thus, the contractile-agonist-dependent differential effects are not explained by changes in the global levels of phosphorylated PKA substrates, but reflected in the regulation of pMLC20 (cross bridge cycling) and F/G actin ratio (actin cytoskeleton integrity, force transmission), implicating a role for compartmentalized signaling involving muscarinic, histamine, and EP receptor subtypes.