We previously showed that peroxisome proliferator-activated receptor delta (PPARdelta) is crucial for embryo implantation as a receptor for cyclooxygenase-2-derived prostacyclin in mice. PPARs belong to the nuclear receptor superfamily. They form heterodimer with a retinoid X receptor, recruit transcriptional cofactors, and bind to a specific recognition element for regulation of target genes. Although cofactors are generally shared by various nuclear receptors, some are involved in cell-specific events. The objective of this investigation was to examine interactions of transcriptional cofactors with PPARdelta in uterine cells for its effectiveness in regulating gene expression. We chose two uterine cellular systems: periimplantation mouse uterus and AN(3)CA human uterine cell line. As examined by in situ hybridization, steroid receptor coactivator (SRC)-2, SRC-3, PPAR-interacting protein, receptor-interacting protein 140 (RIP140), nuclear receptor corepressor (N-CoR), and silencing mediator for retinoid and thyroid hormone receptor (SMRT) exhibit overlapping expression with that of PPARdelta in the periimplantation mouse uterus. Glutathione-S-transferase (GST) pull-down assays show that PPARdelta physically interacts with SRC 1-3, RIP140, PPAR-binding protein, N-CoR, and SMRT in the absence of ligands, suggesting their potent interactions with PPARdelta. Transient transfection assays in AN(3)CA cells show that among members of the SRC family, only SRC-2 serves as a true coactivator for PPARdelta, whereas all SRC members could enhance PPARalpha-induced transcriptional activation. Interestingly, N-CoR and SMRT potently repress PPARdelta-induced transcriptional activation but fail to repress PPARalpha activity. RIP140 is effective in repressing basal and PPAR-induced transcriptional activation. Collectively, the results suggest that gene regulation by PPARdelta in the uterine cells uniquely responds to SRC-2, N-CoR, SMRT, or RIP140, and these interactions may be operative during implantation when these cofactors are abundantly expressed.