Labor occurs primarily in the night/morning hours with initiation peaking between midnight and 5 am. Melatonin (MEL), a pineal gland hormone, acts during the nocturnal phase of the light-dark cycle and increases expression of myometrial contraction-associated proteins that mark the transition of the myometrium from a quiescent to activated state. Previous studies have shown that MEL targets 2 receptors expressed on the human myometrium (MEL receptor 1 and MEL receptor 2 [MT2R]). Other studies have found striking similarities between MEL-regulated expression and oxytocin (OT)-regulated expression of the oxytocin receptor. This study investigated the effects of MEL on myometrial contractility in telomerase-immortalized human myometrial (hTERT) cells expressing oxytocin receptors that were exposed to MEL alone or together with OT. The expression of MT2R was determined in myometrial biopsies from term pregnant women with or without labor and in hTERT cells in vitro. Myometrial cell contractility was measured in the cells with collagen disk retraction assays. The effect of MEL on expression of the gap junction protein, connexin 43, was also investigated. MEL bound to myometrial MT2R synergistically enhanced OT-induced sensitivity and contractility in vitro via phosphorylation of the myosin light chain protein by a mechanism mediated through protein kinase C. Co-treatment of cultured hTERT cells with MEL and OT together increased contractility by 2-fold compared with treatment with OT alone. In tissue samples from laboring patients, there was also a marked elevation in MT2R expression in comparison with matched nonlaboring pregnant women. MEL treatment of the hTERT cells also increased expression of connexin 43. Yellow dye migration assays showed that treatment of hTERT cells with MEL was associated with formation of additional gap junctions. Enhanced expression of connexin 43 and increased levels of cell coupling were mediated through MT2R signaling in a protein kinase C-dependent manner. These findings suggest that MEL acts synergistically with OT to enhance nocturnal uterine contractility at term by direct effects on the contractile machinery and by increased gap junction-mediated intercellular communication. Such synergy between MEL and OT seem to be essential for coordinated and forceful contractions of the late term pregnant uterus and parturition.