Pentafluorophenyl esters of 5- and 6-carboxyfluorescein-3′,6′- O-dipivalate can be easily separated in multigram quantities by column chromatography. The individual isomers were converted into stable phosphoramidites suitable for oligonucleotide synthesis. The use of the cyclohexylcarbonyl (Chc) protecting group instead of pivaloyl (Piv) facilitates the separation of isomers. The fluorescence spectra of 5- and 6-carboxyfluoresceins on oligonucleotides were compared. © 2007 American Chemical Society.