We have previously reported that endometrial expression of matrix metalloproteinase (MMP)-26 mRNA comes to a maximum in the early secretory phase. Since tissue inhibitor of metalloproteinase (TIMP)-4 is a potent inhibitor of MMP-26, the objective of this study was to identify the pattern of TIMP-4 mRNA expression in the normal endometrial cycle. We also evaluated hyperplastic, pre-malignant (atypical hyperplasia) and malignant endometrial tissue. Endometrial TIMP-4 mRNA was localized in tissue sections using in situ hybridization, and quantified in tissue extracts using real-time PCR. Estrogen receptor alpha (ERalpha) was assayed in the same set of samples using immunohistochemistry. In situ hybridization demonstrated TIMP-4 mRNA in the stroma of both normal and pathological tissues. TIMP-4 mRNA increased in the proliferative phase to a maximum in the early secretory phase, and then decreased in the late part of the cycle. Expression was comparable in normal and hyperplastic (including atypical) endometrial samples, whereas lower levels were detected in malignant tumours. Since this general pattern of expression suggests estrogen dependence, we evaluated ERalpha in our samples. Tissue sections from the normal proliferative phase, hyperplasia and pre-malignant atypical hyperplasia tissue stained strongly for ERalpha, whereas weak staining was seen in the secretory phase and in malignant tumours. Thus, low level of ERalpha was accompanied by down-regulated TIMP-4 mRNA, supporting the hypothesis that ERalpha contributes to regulation of the TIMP-4 gene. In addition, we identified a putative estrogen response element (ERE) in the promoter region of the TIMP-4 gene at position -930 to -916. Similarities in the cyclic patterns of TIMP-4 mRNA and MMP-26 mRNA, together with the fact that TIMP-4 is a potent inhibitor of MMP-26, suggest a functional relationship, and furthermore a role in human implantation.