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Members of the 14-3-3 family are intracellular dimeric phosphoserine-binding proteins that can associate with and modulate the activities of many proteins. In our efforts to isolate the genes regulated by progesterone (P4) using suppressive subtractive hybridization, we previously found that 14-3-3t is one of the genes upregulated by P4. In this study, we demonstrated by quantitative RT-PCR (qRT-PCR), western blot analyses, and immunohistochemistry that 14-3-3t mRNA and protein levels were increased in the rat uterus after P4 treatment. Furthermore, qRT-PCR indicated that P4 increased 14-3-3t mRNA levels in human endometrial epithelial cells and endometrial stromal cells (ESCs). Western blot and qRT-PCR analyses revealed that in vitro decidualization using cAMP and medroxyprogesterone 17-acetate increased levels of 14-3-3t mRNA and protein in ESCs. We have shown by qRT-PCR and western blot analyses thatP4 increased themRNAand protein levels of 14-3-3t in Ishikawa cells that stably expressP4 receptor-B (PR-B). Immunocytochemistry revealed that 14-3-3t colocalizes with PR and translocates from the cytoplasm to the nucleus in response toP4.Moreover, by luciferase reporter assay,we demonstrated that 14-3-3t enhances the transcriptional activity of PR-B. Taken together, we propose that 14-3-3t is a P4-responsive gene in uterine cells that modulates P4 signaling. [ABSTRACT FROM AUTHOR]