Red blood cells (RBCs) parasitized by Plasmodium falciparum are rigid and poorly deformable and show abnormal circulatory behavior. During parasite development, knob-associated histidine-rich protein (KAHRP) and P falciparum erythrocyte membrane protein 3 (PfEMP3) are exported from the parasite and interact with the RBC membrane skeleton. Using micropipette aspiration, the membrane shear elastic modulus of RBCs infected with transgenic parasites (with kahrp or pfemp3 genes deleted) was measured to determine the contribution of these proteins to the increased rigidity of parasitized RBCs (PRBCs). In the absence of either protein, the level of membrane rigidification was significantly less than that caused by the normal parental parasite clone. KAHRP had a significantly greater effect on rigidification than PfEMP3, contributing approximately 51% of the overall increase that occurs in PRBCs compared to 15% for PfEMP3. This study provides the first quantitative information on the contribution of specific parasite proteins to altered mechanical properties of PRBCs.