Excessive sequestration of Plasmodium falciparum-infected (pRBC) and uninfected erythrocytes (RBC) in the microvasculature, cytoadherence, and rosetting, have been suggested to be correlated with the development of cerebral malaria. P. falciparum erythrocyte membrane protein-1 (PfEMP1) is the parasite-derived adhesin which mediates rosetting. Herein we show that serum proteins are crucial for the rosette formation of four strains of parasites (FCR3S1, TM284, TM180, and R29), whereas the rosettes of a fifth strain (DD2) are serum independent. Some parasites, e.g., FCR3S1, can be depleted of all rosettes by washes in heparin and Na citrate and none of the rosettes remain when the parasite is grown in foetal calf serum or ALBUMAX. Rosettes of other parasites are less sensitive; e.g., 20% of TM180 and R29 and 70% of TM284 rosettes still prevail after cultivation. A serum fraction generated by ion-exchange chromatography and poly-ethylene-glycol precipitation restored 50% of FCR3S1 and approx 40 to 100% of TM180 rosettes. In FCR3S1, antibodies to fibrinogen reverted the effect of the serum fraction and stained fibrinogen bound to the pRBC surface in transmission electron microscopy. Normal, nonimmune IgM and/or IgG was also found attached to the pRBC of the four serum-dependent strains as seen by surface immunofluorescens. Our results suggest that serum proteins, known to participate in rouleaux formation of normal erythrocytes, produce stable rosettes in conjunction with the recently identified parasite-derived rosetting ligand PfEMP1. Copyright 1999 Academic Press.