We present an efficient methodology to prepare maleimide-tethered, water-soluble gold nanoparticles (maleimide-AuNPs). The maleimide-AuNPs were prepared in the protected form and are readily recovered via a retro-Diels-Alder reaction. The maleimide-AuNPs were fully characterized by H-1 NMR, TGA, TEM, and XPS and were determined to have a gold core with an average size of 3.2 +/- 0.8 nm; each core contains about 1000 gold atoms and is surrounded by 30 maleimide-terminated ligands and 60 thiolated PEG ligands. The maleimide-AuNPs efficiently react with rhodamine 123 and cysteine and are a promising template for biological applications.