We have previously reported that endometrial mRNA expression of both tissue inhibitors of metalloproteinase-4 (TIMP-4) and matrix metalloproteinase-26 (MMP-26) peaks in the early secretory phase, which implies a role in implantation. The objective of this study was to compare the distribution of TIMP-4 and MMP-26 in endometrial tissue and uterine fluid over the menstrual cycle. Endometrial tissue was analysed with in situ hybridization and immunohistochemistry to localize mRNA and protein for TIMP-4 and MMP-26 in the same set of samples. TIMP-4 mRNA was quantified in separated stromal and epithelial cells using real-time PCR. Uterine fluid was analysed with western blotting. TIMP-4 mRNA was exclusively localized to the stroma, whereas MMP-26 mRNA was expressed by epithelial cells. TIMP-4 protein was only occasionally found in the stroma but was consistently present in granules of the apical part of luminal and glandular epithelial cells. TIMP-4, but not MMP-26, was demonstrated in uterine fluid. Thus, TIMP-4 is produced in the stroma only, secreted by stromal cells, taken up by epithelial cells, accumulated in apical granules and finally secreted to the uterine fluid. Maximal expression of MMP-26, and its strongest inhibitor TIMP-4, in the early and mid-secretory phase suggests a role during implantation. MMP-26 is stored in epithelial cells in its active form, is not released spontaneously and is controlled by TIMP-4 in both stroma and uterine fluid.