A sensitive high-performance liquid chromatography (HPLC) method has been developed for the determination of bound sulfur in mammalian serum. In this work, bound sulfur is defined as divalent sulfur that is easily liberated as sulfide by reduction with dithiothreitol. Released sulfide was treated by flow gas dialysis and converted into a fluorescent derivative, thionine, through the reaction with p-phenylenediamine and ferric ion. Thionine was then determined by HPLC using fluorometric detection. The calibration curve for bound sulfur is linear in the range from 0.1 to (10 \mu \mathrm{M}), and the sensitivity of this method allows detection of less than (10 \mathrm{~nm}) bound sulfur ( (0.5-\mathrm{ml}) sample). Recoveries of low-molecular-weight and high-molecular-weight bound sulfur added to human serum at (1.0-5.0 \mu \mathrm{M}) levels averaged between 94 and (101 %). The proposed method was applied to the determination of bound sulfur in human and various animal sera. The mean concentration in normal human serum was (1.16 \pm 0.09 \mu \mathrm{M}) for males ((n=5)) and (1.07 \pm 0.18 \mu \mathrm{M}) for females ((n=5)). The mean concentrations ranged from 1.55 to (6.18 \mu \mathrm{M}) in animal sera. The form of the sulfur bound in serum was also discussed. 1993 Academic Press, Inc.