The flagellar calcium-binding protein (FCaBP) of Trypanosoma cruzi is localized to the flagellar membrane in all life cycle stages of the parasite. Myristoylation and palmitoylation of the N terminus of FCaBP are necessary for flagellar membrane targeting. Not all dually acylated proteins in T. cruzi are flagellar, however. Other determinants of FCaBP therefore likely contribute to flagellar specificity. We generated T. cruzi transfectants expressing the N-terminal 24 or 12 amino acids of FCaBP fused to GFP. Analysis of these mutants revealed that although amino acids 1-12 are sufficient for dual acylation and membrane binding, amino acids 13-24 are required for flagellar specificity and lipid raft association. Mutagenesis of several conserved lysine residues in the latter peptide demonstrated that these residues are essential for flagellar targeting and lipid raft association. Finally, FCaBP was expressed in the protozoan Leishmania amazonensis, which lacks FCaBP. The flagellar localization and membrane association of FCaBP in L. amazonensis suggest that the mechanisms for flagellar targeting, including a specific palmitoyl acyltransferase, are conserved in this organism.