The mechanisms by which proteins are targeted to flagella and cilia are poorly understood. We set out to determine the basis for the specific localization of a 24 kDa flagellar calcium-binding protein (FCaBP) expressed in all life cycle stages of Trypanosoma cruzi. Through the study of trypanosome transfectants expressing various FCaBP deletion mutants, we found that the N-terminal 24 amino acids of the protein are necessary and sufficient for flagellar localization. Subsequent experiments revealed that FCaBP is myristoylated and palmitoylated and, in fact, is one of very few proteins in the cell possessing these acyl modifications. Both fatty acids are required for flagellar localization, suggesting that FCaBP localization may be mediated through association with the flagellar plasma membrane. Indeed, FCaBP associates with the flagellar membrane in a calcium-dependent manner, reminiscent of the recoverin family of calcium-myristoyl switch proteins. Thus, FCaBP is a novel member of the calcium-acyl switch protein family and is the only member described to date that requires two fatty acid modifications for specific membrane association. Its unique localization mechanism is the first described for any flagellar protein. The existence of such a protein in this protozoan suggests that acylation and calcium switch mechanisms for regulated membrane association are conserved among eukaryotes.