Several chiral stationary phases (CSPs) were examined to separate the enantiomers of A-69992, a chiral HIV anti-infective nucleoside. The only CSP found to be effective was Nucleosil Chiral-1, a ligand-exchange CSP, which was used to prepare microgram amounts of the enantiomers of high optical purity. This appears to be the first separation of the enantiomers of a nucleoside by chiral high-performance liquid chromatography.