A Giardia-specific protein family denominated as ␣-giardins, represents the major protein component, besides tubulin, of the cytoskeleton of the human pathogenic parasite Giardia lamblia. One of its members, ␣19-giardin, carries an N-terminal sequence extension of MGCXXS, which in many proteins serves as a target for dual lipid conjugation: myristoylation at the glycine residue after removal of the methionine and palmitoylation at the cysteine residue. As the first experimental evidence of a lipid modification, we found ␣19-giardin to be associated with the membrane fraction of disrupted trophozoites. After heterologous coexpression of ␣19-giardin with giardial N-myristoyltransferase (NMT) in Escherichia coli, we found the protein in a myristoylated form. Additionally, after heterologous expression together with the palmitoyl transferase Pfa3 in Saccharomyces cerevisiae, ␣19-giardin associates with the membrane of the main vacuole. Immunocytochemical colocalization studies on wild-type Giardia trophozoites with tubulin provide evidence that ␣19-giardin exclusively localizes to the ventral pair of the giardial flagella. A mutant in which the putatively myristoylated N-terminal glycine residue was replaced by alanine lost this specific localization. Our findings suggest that the dual lipidation of ␣19-giardin is responsible for its specific flagellar localization.