Several series of 14-membered ketolides derived from erythromycin exhibit useful antimicrobial activity against macrolide-resistant bacteria. To determine if 16-membered ketolides may possess analogous activity, 3-keto derivatives of 5-O-mycaminosyl-23-O-acetyltylonolide and desmycosin were synthesized by protection of susceptible functional groups, oxidation of the 3-hydroxyl group under modified Moffatt-Pfitzner conditions, and subsequent deprotection. The resulting 3-keto products unexpectedly adopted the 2, 3-trans enol rather than the 3-keto tautomer. The trans configuration of the 2, 3-double bond in the macrolide chain is most likely the result of hydrogen bond stabilization between the enol hydroxyl and lactone carbonyl, which places these two groups in a cis relationship. This preference for the enol tautomer in 16-membered macrolides is not seen with 14-membered ketolides. The in vitro antimicrobial activity of the enol derivatives was greatly reduced compared to their unoxidized parent compounds, but the reduced antimicrobial activity of the enol derivatives paralleled results from corresponding 2, 3-anhydro derivatives of 16-membered macrolides, which also have 2, 3-trans stereochemistry. These results are in contrast to those from 14-membered-ring macrolides in which 3-keto and 2, 3-anhydro derivatives exhibit greater activity than 3-hydroxy compounds.