Introduction: Prostaglandin F2α (PGF2α) is a myometrial stimulant, both PGF2α and its receptor are reported to increase towards parturition. The mechanism by which PGF2α exerts its affects on uterine excitation-contraction coupling is unknown. We examined PGF2α effects on Ca2+ signalling and force in pregnant rat myometrium. Methods: PGF2α was examined on both longitudinal strips and isolated myometrial cells from pregnant Wistar rats. Strips were loaded with the calcium sensitive indicator Indo-1AM and simultaneous recording of [Ca2+]i and force were made using a photometric system combined with force measurements. Cells were isolated using Liberase Blendzyme 3 (Roche), loaded with Fluo-4AM, and [Ca2+]i recorded using Nipkow Disk based confocal imaging system. Data were analysed using T-tests, and results shown as mean ± SEM. Results: Application of PGF2α on myometrial strips in the presence of external Ca2+ initiated bursts of Ca2+ spikes associated with phasic contractions which were fully abolished in Ca2+ free solution (n=5). In the absence of external Ca2+, PGF2α produced a transient increase in [Ca2+]i 2.74 ±0.8 and force 9.00±3.2 expressed as a percentage of high-K+ induced response taken as 100% (n=6, p=0.002), Ca2+ appeared as propagating Ca2+ waves. Application of PGF2α in the absence of external Ca2+ resulted in the depletion of the sarcoplasmic reticulum (SR) and re-admission of external Ca2+ following SR Ca2+ depletion resulted in a sustained rise in basal [Ca2+]i to 94.35 ±0.8% of high-K+ (n=6), and activation of Ca2+ bursts superimposing the raised [Ca2+]i. Ca2+ was associated with activation of phasic contractions superimposed on tonic force. Nifedipine (L-type Ca2+ channel blocker) caused inhibition of the busts of Ca2+ spikes and phasic contractions, but had little effect on the sustained component of both Ca2+ and force. Basal [Ca2+]i and tonic force in the presence of nifedipine were: 87.11±10.7 and 65.71±15.5% of high-K+ (n=5, p=0.25). The raised [Ca2+]i and tonic force were significantly reduced by La3+ (non-specific blocker of store operated Ca2+ entry (SOCE)), [Ca2+]i was reduced from 94.35±0.8, to 91.54±1.4% of high-K+ (n=5, p=0.05). Conclusion: PGF2α caused release from the SR which appeared as a propagating Ca2+ wave causing depletion of the store. Re-admission of external Ca2+ resulted in activation of a nifedipine-resistance Ca2+ influx sensitive to La3+. This suggests that the stimulant action of PGF2α is associated with activation of a Ca2+-release Ca2+-entry coupling mechanism leading to opening of the SOCE pathway.