Abstract Methods are described for the synthesis of [ 3H]chloramphenicol and derivatives labeled on carbon 1 of the propanediol side chain, with a specific activity of about 2 mCi/μmol. The labeling step involves the reduction of the I-oxo derivative of N-acetyl chloramphenicol base by KB 3H 4 to produce a mixture of the d (−) threo- and d (−) erythro-diastereoisomers, since carbon 1 is an asymmetric carbon atom. The two isomers were separated by thin-layer chromatography after acetylation of the two free hydroxyls. After hydrolysis of the three acetyl groups, the biologically active d (−) threo-[1 − 3H]chloramphenicol base was converted to chloramphenicol. Modification of the above procedures allows the rapid and simple preparation of the mixed d (−) threo- and d (−) erythro-isomers of [1 − 3H]chloramphenicol. This mixture can be used where the presence of the inactive d (−) erythro-isomer of chloramphenicol is not important. The modified procedure also allows the preparation of the mixed isomers of [1 − 3H]chloramphenicol base and of chloramphenicol analogs. Attempts to prepare a 3-aldehyde derivative of chloramphenicol were not successful. If this could be done, reduction of this derivative with KB 3H 4 would produce the correct isomer of chloramphenicol since carbon atom 3 on the propanediol side chain is not an asymmetric carbon atom.