Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a membrane-bound serine proteinase inhibitor having two extracellular Kunitz-type proteinase inhibitor domains (KD) namely KD-1 and KD-2. It efficiently inhibits hepatocyte growth factor activator, matriptase, hepsin, prostasin and trypsin. We have previously reported that the expression of HAI-1 suppresses the in vitro invasive capability of human glioblastoma cells. In this study we examined the role of each KD in the anti-invasive effect of HAI-1. Engineered over-expression of the mature membrane-form HAI-1 suppressed in vitro fibrin gel invasion of two human glioblastoma cell lines, U251 and YKG-1. The migratory activity on type IV collagen was also suppressed by the HAI-1 expression. These effects were not affected by the deletion of intracytoplasmic domain of HAI-1. A truncated secreted form of HAI-1 also suppressed in vitro invasion of the cells, indicating that the extracellular portion of HAI-1 was responsible for the anti-invasive effect. To determine the roles of each KD in the anti-invasive effect of HAI-1 in vitro, we constructed expression plasmids for HAI-1 with or without mutation at the P1 position of the reactive site of each KD. The results revealed that the proteinase inhibitor activity of N-terminal KD (KD-1) is responsible for the anti-invasion effect of HAI-1.