The hypothesis that phosphorylation of progesterone receptor (PR) isoforms, PR-A and PR-B, in myometrial cells affects progesterone action in the context of human parturition was tested. Immunodetection of phosphoserine (pSer) PR forms in term myometrium revealed that the onset of labor is associated with increased phosphorylation of PR-A at serine-345 (pSer345-PRA) and that pSer345-PRA localized to the nucleus of myometrial cells. In explant cultures of term myometrium generation of pSer345-PRA was induced by interleukin-1β and dependent on progesterone, suggesting that pSer345-PRA generation is induced by a proinflammatory stimulus. In the hTERT-HMA/B human myometrial cell line, abundance of pSer345-PRA was induced by progesterone in a dose- (EC50 ∼1 nM) and time-dependent manner. Prevention of pSer345 (by site-directed mutagenesis) abolished the capacity for PR-A to inhibit anti-inflammatory actions of progesterone mediated by PR-B but had no effect on the transrepressive activity of PR-A at a canonical progesterone response element. Taken together, the data show that human parturition involves the phosphorylation of PR-A at serine-345 in myometrial cells and that this process is ligand dependent and induced by a proinflammatory stimulus. We also found that in myometrial cells, pSer345 activates the capacity for PR-A to inhibit antiinflammatory actions of progesterone mediated by PR-B. Phosphorylation of PR-A at serine-345 may be an important functional link between tissue-level inflammation and PR-A-mediated functional progesterone withdrawal to trigger parturition.