Intercellular communications through gap-junctions are crucial for synchronising myometrial activity but the mechanisms of these communications in native tissue remain poorly elucidated. Cultured uterine myocytes, often used in research are a convenient, but not ideal model of human myometrium. Studying intercellular communications between uterine myocytes in ultra-thin myometrial slices presents the advantage of preserving the intercellular contacts within the tissue in its native state. In this study, we investigated the effect of mechanical stretch on evolution of intercellular communications in human myometrium. Changes in cytoplasmic Ca2+ elicited by spontaneous action potentials and applications of oxytocin (OT, 100 nM) were recorded daily from slices maintained in organotypic culture for one week. Myometrial biopsies were taken from patients undergoing Caesarean Section with informed written consent and approval from the Local Ethics Committee (REC-05/Q2802/107). The slices, 200 μm thick, were cut using a vibroslicer in modified Krebs solution (4°C) and cultured in SmGM-2 medium (Cambrex) with 5% foetal calf serum. Ca2+ signalling events were recorded from Fluo-4 loaded slices using a Zeiss LSM 510 META confocal microscope at 1 frame/s. Myometrial slices were cultured under two different conditions, unstretched, free floating slices and stretched slices(slices were pinned to the bottom of the Sylgard-filled Petri dish at 150% of their slack length). Spontaneous activity was observed in all freshly cut slices. After one day in culture, spontaneous activity was observed only in 1 out of 11 unstretched slices and in 2 out of 10 stretched slices. No spontaneous activity was observed after two or more days of culturing. In freshly cut slices, application of OT triggered synchronous Ca2+ transient in all cells within the field of view. The synchronicity of the Ca2+ transients was quantified as standard deviation of time delays between the oxytocin application and peaks of Ca2+ transients in different cells within the field. During culturing, there was a marked decrease in synchrony in both stretched and unstretched slices. That is, the standard deviations of the Ca2+ transient time delays in unstretched and stretched slices increased, respectively, from 8.2 s and 7.4 s in freshly cut slices to 30.6 s and 41.9 s after 7 days in culture. Upon culturing and loss of synchrony, some cells demonstrated spontaneous calcium oscillations that were unaffected by OT and did not propagate to neighbouring cells. In conclusion, our data show that the loss of spontaneous activity and intercellular coupling in human myometrium under culture conditions could be delayed but not prevented by mechanical stretch.