BACKGROUND: Individually, the red blood cell (RBC) polymorphisms sickle cell trait (HbAS) and alpha+thalassemia protect against severe Plasmodium falciparum malaria. It has been shown through epidemiological studies that the co-inheritance of both conditions results in a loss of the protection afforded by each, but the biological mechanisms remain unknown. METHODS: We used RBCs from >300 donors of various HbAS and alpha+thalassemia genotype combinations to study the individual and combinatorial effects of these polymorphisms on a range of putative P. falciparum virulence phenotypes in-vitro, using four well-characterised P. falciparum laboratory strains. We studied cytoadhesion of parasitized RBCs (pRBCs) to the endothelial receptors CD36 and ICAM1, rosetting of pRBCs with uninfected RBCs, and pRBC surface expression of the parasite-derived adhesion molecule P. falciparum Erythrocyte Membrane Protein-1 (PfEMP1). FINDINGS: We confirmed previous reports that HbAS pRBCs show reduced cytoadhesion, rosetting and PfEMP1 expression levels compared to normal pRBC controls. Furthermore, we found that co-inheritance of HbAS with alpha+thalassemia consistently reversed these effects, such that pRBCs of mixed genotype showed levels of cytoadhesion, rosetting and PfEMP1 expression indistinguishable from those seen in normal pRBCs. However, pRBCs with alpha+thalassemia alone showed parasite strain-specific effects on adhesion, and no consistent reduction in PfEMP1 expression. INTERPRETATION: Our data support the hypothesis that the negative epistasis between HbAS and alpha+thalassemia observed in epidemiological studies might be explained by host genotype-specific changes in the pRBC-adhesion properties that contribute to parasite sequestration and disease pathogenesis in vivo. The mechanism by which alpha+thalassemia on its own protects against severe malaria remains unresolved.