When chloramphenicol was incubated with rat liver microsomes, four previously unidentified metabolites were detected and identified. They include chloramphenicol aldehyde (chloramphenicol with the primary alcohol group oxidized to an aldehyde group), p-nitro-benzyl alcohol, N-(2-oxoethyl)dichloroacetamide, and N-(2-hydroxyethyl)dichloroacetamide. The formation of these metabolites was dependent upon the presence of NADPH and O2 and was inhibited when SKF 525-A or CO/O2 (8:2, v/v) were present in the reaction mixture. Moreover, the metabolites were formed by liver microsomes from phenobarbital-treated rats but not by microsomes from untreated rats or rats treated with beta-naphthoflavone. The formation of these metabolites is consistent with a mechanism that involves an initial oxidation of chloramphenicol to chloramphenicol aldehyde by cytochrome P-450. Inasmuch as this metabolite is a beta-hydroxyaldehyde, it can chemically undergo a retro-aldol cleavage to p-nitrobenzaldehyde and N-(2-oxoethyl)dichloroacetamide. Enzymatic reduction of these aldehyde intermediates would yield p-nitrobenzyl alcohol and N-(2-hydroxyethyl)dichloroacetamide, respectively.