A high-performance liquid chromatography method with on-line radioactivity monitoring was developed for the measurement of 2 -fluoro-2-deoxy-D-[U- (\left.{ }^{14} \mathrm{C}\right]-) glucose-derived metabolites in a cell culture system of human chondrocytes embedded in soft agarose. To optimize the chromatographic procedure, glucose-analogous substrates derived from 2 -fluoro-2-deoxy-D-glucose by enzymatic synthesis in vitro were used. The synthesized metabolites could be separated by anionexchange chromatography on a Partisil 10 SAX cartridge with a LiChrosorb RP 18-5 guard column eluted with a (35-\mathrm{min}) ion-strength/pH gradient performed from (15 \mathrm{mM}{\mathrm{NH}}^{4} \mathrm{H}{2} \mathrm{PO}{4}, \mathrm{pH} 3.8), to (0.75 \mathrm{M} \mathrm{NH}{4} \mathrm{H}{2} \mathrm{PO}{4}), pH 4.8 , at a fow rate of (2 \mathrm{ml} / \mathrm{min}). Only by using an on-line radioactivity monitor instead of an off-line counting procedure was the resolution obtained sufficient for the determination of these intermediates. This method was applied to studying the metabolic pathway of 2-fluoro-2-deoxy-D-glucose in human chondrocytes. Due to the resistance of the chondrocytes embedded in soft agarose, the usual cell-lysing methods could not be used; therefore, an extraction procedure for acid-stable glucose metabolites, which may also be applied to other resistant cell lines or critical cell culture systems, was developed. With the procedure presented here, the existence of metabolites of 2-fluoro-2-deoxy-D-glucose resulting from enzymatic reactions following the hexokinase reaction could be proven. We present here for the first time evidence that chondrocytes are able to metabolize 2-fluoro-2-deoxy-D-glucose to the UDP-activated sugars UDP-2-fluoro-2-deoxy-D-glucose, UDP-2fuoro-2-deoxy-D-galactose, and UDP-2-fluoro-2deoxy-D-glucuronic acid. ๔ 1993 Academic Press, Inc.