The extracellular matrix proteolytic machinery has long been recognized as one of the most important mechanisms for regulating trophoblast invasion. Matrix metalloproteinases (MMPs) are a group of proteases involved in this process, and their activities are regulated by tissue inhibitors of MMPs (TIMPs). In this study, we collected rhesus monkey uteri on Days 12, 18, and 26 of pregnancy and examined the mRNA expression of MMP-2, -9, -14, and TIMP-1, -2, -3, as well as the activities of MMP-2 and -9 by using in situ hybridization and gelatin zymography, respectively. The results showed that MMP-2 and -9 were expressed earlier than MMP-14 and TIMPs in the pregnant endometrium. MMP-14 and TIMP-2 mRNAs appeared in perivascular decidual cells earlier than MMP-2 mRNA. On Day 26 of pregnancy, placental villi expressed little MMP-2, -14, and TIMP transcripts but abundant MMP-9 mRNA. Furthermore, MMP-2, -9, -14, and TIMP-1, -2, -3 were highly expressed on the fetal-maternal border but were absent in the myometrium. TIMP-3 mRNA in the endometrium was specifically localized to some cells lining the outer membrane of several groups of arterioles. Combined with the results obtained by gelatin zymography, we found that active MMP-2 existed in the endometrium throughout these three phases, while MMP-9 showed considerable activities only on Days 18 and 26 of pregnancy. The data suggest key roles for MMP-2 and -9 in invasion of trophoblast cells into the endometrium and the development of the placenta and might indicate that these processes are regulated by MMP-14 and TIMP-1, -2, and -3.