Geldanamycin (GA), an antibiotic of the ansamycin family and an inhibitor of heat shock protein 90 (Hsp90), was previously shown to inhibit the malarial parasite, Plasmodium falciparum. Here we report that cyclosporin A (CsA), an inhibitor of parasitic cyclophilin (Cyp) and protein phosphatase 2B (calcineurin, CN), acted synergistically with GA to inhibit the erythrocytic growth of the parasite. Parasitic calcineurin associated with Hsp90 in vivo, and GA inhibited the association, but CsA had no effect. In a number of CsA-resistant (CsA(R)) P. falciparum clones mutations were detected in functionally significant amino acid residues of the catalytic and regulatory subunits of calcineurin (CnA and CnB, respectively) and in two out of three parasitic cyclophilins, namely Cyp19A and Cyp19B. No mutation was detected in the third cyclophilin, Cyp24. Further analysis of the mutant CnA revealed that its protein phosphatase activity was highly CsA-resistant in vitro. Similarly, one of the mutant Cyp19A proteins was purified and found to be unable to inhibit parasitic CN in the presence of CsA. Together, these results underscore the importance of the proper assembly and function of CN in plasmodial biology and suggest that the inhibition of CN can be a potential mechanism behind the CsA-sensitivity of the malaria parasite.