OBJECTIVE: The purpose of this study was to enrich vasoactive factors that are present in the plasma of women with preeclampsia by the application of sequential fractionation and determination of the activity of each fraction in a bioassay. STUDY DESIGN: Pooled plasma from women with preeclampsia (n = 23) and matched control subjects (n = 23) was subjected to fractionation with ultrafiltration, targeted immunodepletion, or size exclusion chromatography. Myometrial arteries that were isolated from healthy cesarean section biopsy specimens (n = 28) were incubated with plasma fractions (2%, volume/volume), and their endothelial function was assessed by wire myography. RESULTS: Incubation of arteries with preeclampsia plasma or immunodepleted preeclampsia plasma had a deleterious effect on endothelial-dependent relaxation. Bioactivity of the plasma factors was absent in all fractions after either plasma ultrafiltration or separation with the use of size exclusion chromatography; however, activity was restored after recombination of these fractions. CONCLUSION: This study provides the first conclusive evidence that multiple synergistic factors, with a combined vasoactive effect, are present in the plasma of women with preeclampsia.