Dietary and pharmacologic isothiocyanates (ITCs) may play a role in reducing the risk of certain cancers. The quantification of ITCs in humans is important both for epidemiological and pharmacokinetic studies. We describe a modification of an HPLC-based assay of urinary ITCs for use with human plasma. The assay utilizes the cyclocondensation reaction of 1,2-benzenedithiol with ITCs present in human plasma, followed by a twostep hexane extraction and analysis by HPLC using UV detection at 365 nm. The method shows linearity and reproducibility with human plasma over a range of 49 -3003 nM phenethyl isothiocyanate (PEITC) (r 2 ؍ 0.996 ؎ 0.003). A similar degree of linearity was seen with two other biologically occurring conjugates of PEITC: PEITC-N-acetylcysteine (PEITC-NAC) and PEITC-glutathione (PEITC-GSH). The recovery of PEITC assessed on multiple days was 96.6 ؎ 1.5% and was 100% for PEITC-GSH and PEITC-NAC. The reproducibility of the assay on multiday samplings showed a mean %CV of 6.5 ؎ 0.3% for PEITC, 6.4 ؎ 4.3 for PEITC-NAC and 12.3 ؎ 3.9 for PEITC-GSH. In clinical studies, mean plasma ITC level of 413 ؎ 193 nM PEITC equivalents was determined for a non-dietary-controlled group of 23 subjects. Multiday analysis data from pharmacokinetic plasma sets of 3 subjects taking a single dose of PEITC at 40 mg showed a good CV (range: 16 -21%). The applicability of the methodology to pharmacokinetic studies of PEITC in humans is demonstrated.