The human endometrium is a dynamic tissue that undergoes cyclic changes under the influence of steroid hormones as well as numerous local paracrine and autocrine factors. Heat shock 70 kDa protein (HSPA5; also known as GRP78/BiP), a molecular chaperone within the endoplasmic reticulum, plays crucial roles in normal cellular processes as well as in stress conditions, in which it is a central regulator for the unfolded protein response (UPR). We hypothesized that HSPA5 expression level is variable throughout the menstrual cycle in human endometrium and that estrogen signaling cross-talks with UPR signaling by interacting with HSPA5. HSPA5 expression throughout the menstrual cycle was evaluated in vivo in normal human endometrium. Using in vitro techniques, we then assessed the bidirectional regulation of HSPA5 and estrogen signaling in human endometrial glandular (Ishikawa) and stromal cells (ESC). HSPA5 immunoreactivity in endometrial glandular and stromal cells was cycle-dependent, and was significantly higher in phases of the menstrual cycle when estradiol (E(2)) levels are known to be the lowest compared with the rest of the cycle (P < 0.001). E(2) did not affect HSPA5 expression after 8-24 h incubation in Ishikawa cells and ESC in vitro. However, tunicamycin-induced HSPA5 expression was significantly lowered in these cells when pretreated with E(2) (P < 0.01 and P < 0.05, respectively). On the other hand, tunicamycin decreased E(2) up-regulated alkaline phosphatase activity (P < 0.001). In conclusion, there is cycle-dependent HSPA5 expression with a possible inverse correlation between HSPA5 expression and E(2) levels in human endometrium. We suggest that estrogen signaling cross-talks with the UPR cascade by interacting with HSPA5, as supported by our in vitro findings.