Phosphorylation of components of the erythrocyte membrane skeleton has major effects on the physical properties of the membrane. Infection of red cells by the protozoan parasite Plasmodium falciparum leads to a marked increase in the level of phosphorylation of red cell protein 4.1 and the insertion into the red cell skeleton of parasite-encoded phosphoproteins, including the mature-parasite-infected erythrocyte surface antigen (MESA). Because of the tight association of MESA with protein 4.1, we set out to determine the importance of this interaction and that of other parasite-encoded skeletal-associated proteins to phosphorylation of the infected red cell membrane. Our results show that neither MESA nor protein 4.1 is required for phosphorylation of its binding partner. Further, phosphorylation of MESA and protein 4.1 occurs independently of the presence of knobs, the expression of PfHRP1, or cytoadherence phenotype. In contrast to previous studies, we were unable to detect a change in the molecular weight of protein 4.1 in erythrocytes infected with cytoadherent parasite lines. In red cells infected with parasites expressing PfHRP1 (K+), MESA and protein 4.1 are substrates for a kinase with the inhibitor profile of a casein kinase. Surprisingly, however, when we examined phosphorylation of MESA and protein 4.1 in K(-)-infected erythrocytes, we found that casein kinase I and II inhibitors had no, or greatly reduced, effectiveness, and in fact, phosphorylation of these two proteins was enhanced in some instances.