Matrix metalloproteinases (MMPs) are implicated in the degradation of extracellular matrix; they play important roles in the invasion of the trophoblast cell into the maternal endometrium during placentation. Previous studies have concentrated on comparison of MMP expression in trophoblast cells between the first and third trimester. But the dynamic expression of MMPs during the first trimester has not been reported. In the present study, the expression of MMP-2, -9, and -14 (membrane-type MMP-1) and the production of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) by cultured human cytotrophoblast cells from 6 to 11 wk of gestation were investigated. The cells were cultured under serum-free conditions. There was no MMP-9 secretion by the cells at Week 6, but from Week 7 to 11 the MMP-9 secretion increased gradually. Week 11 cells secreted more than 10-fold as much MMP-9 (167.7 ± 18.8 ng/ml) as Week 7 (14.7 ± 3.9 ng/ml) cultures. However, MMP-2 production declined from Week 6 to Week 11, and the production at Week 11 (32.3 ± 8.1 ng/ml) was about one sixth that at Week 6 (205.7 ± 27.2 ng/ml). The expression of mRNA transcripts for MMP-2 and MMP-9 correlated with enzyme secretion; we did not detect any MMP-9 mRNA signal in 20 μg total RNA extracted from cultured cells at Weeks 6, 7, and 8 of pregnancy, but a signal was apparent in Weeks 9 and 11. MMP-2 mRNA was expressed throughout the 6- to 11-wk period and exhibited a remarkable decline during this period. MMP-14 mRNA transcripts remained relatively stable from 6 to 11 wk. Significantly more TIMP-1 (P < 0.01) was detected in Week 9 (87.5 ± 15.0 ng/ml) and Week 11 (169.1 ± 30.2 ng/ml) media compared to Week 6 media (23.5 ± 4.8 ng/ml), but we did not detect any TIMP-2 in the media of the tested cells. This study demonstrated that first-trimester human cytotrophoblast cells were able to produce abundant laminin, fibronectin, and vitronectin. However, we did not observe detectable secretion of collagen I and collagen IV. These data indicated that human trophoblast-derived MMPs and their inhibitors are intrinsically and developmentally regulated. The same cytotrophoblast cells that produced MMPs could also secrete various substrates for these enzymes.