Protein prenylation, involving the alkylation of a specific C-terminal cysteine with a C-15 or C-20 isoprenoid unit, is an essential posttranslational modification required by most GTP-binding proteins for normal biological activity. Despite the ubiquitous nature of this modification and numerous efforts aimed at inhibiting prenylating enzymes for therapeutic purposes, the function of prenylation remains unclear. To explore the role the isoprenoid plays in mediating protein-protein recognition, we have synthesized a photoactivatable, isoprenoid-containing cysteine analogue (2) designed to act as a mimic of the C-terminus of prenylated proteins. Photolysis experiments with 2 and RhoCDI (GDI), a protein which interacts with prenylated Rho proteins, suggest that the GDI is in direct contact with the isoprenoid moiety. These results, obtained using purified GDI as well as Escherichia coli (E. coli) crude extract containing GDI, suggest that this analogue will be an effective and versatile tool for the investigation of putative isoprenoid binding sites in a variety of systems. Incorporation of this analogue into peptides or proteins should allow for even more specific interactions between the photoactivatable isoprenoid and any number of isoprenoid binding proteins.