Previous studies indicated that calcitonin (CT), a peptide hormone involved in calcium (Ca2+) homeostasis, is transiently induced by steroid hormone progesterone (P) in the uterine epithelia of the rat and human within the window of implantation. Targeted disruption of uterine CT expression markedly impaired implantation in the rat. To gain insight into the molecular events underlying CT action in the endometrium, we performed gene expression profiling in response to CT in a human endometrial adenocarcinoma cell line, Ishikawa. We identified the gene encoding tissue tranglutaminase type II (tTGase), which participates in Ca2+-dependent, protein-protein cross-linking, as a downstream target of CT. Interestingly, addition of P alone to Ishikawa cells led to a marked induction in the level of both CT and tTGase, indicating the existence of a pathway involving P receptors, CT, and tTGase in these cells. Other studies revealed that regulation of the tTGase gene by CT occurs via its cell surface receptor and uses both cAMP and Ca2+ signaling pathways. We also noted that tTGase protein is expressed in human endometrium during the P-dominated midsecretory phase of the menstrual cycle, and it is localized at the basal membrane of glandular epithelium and the surrounding stroma. The spatio-temporal expression of tTGase in human endometrium during the cycle closely overlapped with that of CT. In summary, we have uncovered a novel steroid-regulated signaling cascade in which P induces CT, which, in turn, induces tTGase and potentially plays a critical role in the human endometrium during implantation.