Human endometrium remodels extensively during each reproductive cycle culminating in loss of most functionalis tissue at menstruation. Evidence suggests that menstruation results from the action of the matrix metalloproteinases (MMP), enzymes secreted in latent forms. MMP activation is thus an important regulatory step. It has not been established that MMPs are active within menstrual endometrium in vivo. We used in situ zymography to demonstrate active forms of MMPs in human endometrium across the normal menstrual cycle. Both gelatinase and collagenase activities were detected in most endometrial tissues. Semiquantitation demonstrated a substantial and significant increase in both gelatinase and collagenase activity in menstrual samples compared with those at any other time of the cycle. Gelatinase activity was both associated with cells and extracellular. All collagenase activity was extracellular. Immunoreactive MMP-2 and MMP-9 colocalized with active gelatinase, although much immunoreactive gelatinase was inactive. Some gelatinase activity colocalized with CD45(+) leukocytes. Menstruation is initiated at discrete foci, and active MMPs were similarly at foci within the tissue. This is the first in vivo evidence for increased active MMPs in menstrual endometrium compared with other stages of the cycle. These findings position the MMPs for a critical role in the matrix degradation at menstruation.