Plasminogen activator inhibitor type 1 (PAI1) is considered to be the main regulator of fibrinolytic activity in blood and has been identified as a key-enzyme in the metastasis and vascularization of solid tumors. The aim of this study was to determine whether high or low plasma levels and/or activity of PAI1 correlate with the presence of metastatic disease for patients with melanoma. We hypothesized that the presence of metastases could result in a disturbance of the fibrinolytic balance of the blood. To test our hypothesis, we have developed a unique enzyme-linked immunosorbent assay (ELISA) that can measure both the total amount as well as the active fraction of PAI1 in the plasma. We then used this novel assay to analyze the plasmatic PAI1 levels and activity of patients with advanced melanoma (AM, n = 18) and primary melanoma (PM, n = 21) and compare it to a control population (n = 38). We found no statistically significant difference in the total plasmatic PAI1 levels between the controls and patients with PM or AM (P = 0.6199). In contrast, there was a significant difference in the active fraction of PAI1 between the controls and patients with PM or AM (P = 0.0076). The difference between the control and AM groups was highly significant (P = 0.0042). A value of less than 44% active PAI1 was shown to be clinically meaningful by linear discriminant analysis. Surprisingly, the difference between the control and PM groups was also significant--although borderline (P = 0.0488). Of the patients with PM, 19% had PAI1 activity values less than 44%, which strongly supports further investigations to determine whether plasmatic PAI1 activity might be a biological marker of increased metastatic risk.