Plasminogen activator inhibitor-1 (PAI-1) is up-regulated strongly in various cancer tissues, including colon cancer tissue. Highly specific rabbit polyclonal antibodies against PAI-1 were used for immunohistochemical localization of PAI-1 in 12 invasive colorectal adenocarcinomas. PAI-1 immunoreactivity was observed in endothelial cells of some vessels located in the submucosa and in several fibroblast-like cells located at the invasive front. No PAI-1 immunoreactivity was seen in cancer cells in any of the 12 cases. Double immunofluorescence using the PAI-1 antibodies together with antibodies against alpha-smooth muscle actin for myofibroblast/smooth muscle cells and CD34 for endothelial cells showed that more than 80% of the PAI-1-positive fibroblast-like cells in all 12 cases were myofibroblasts. In 4 of 12 cases, a few of the PAI-1-positive fibroblast-like cells in the invasive front were CD34+. We conclude that the majority of PAI-1-positive fibroblast-like cells located at the leading edge of the invasive colon cancers are myofibroblasts.