A simple high-performance liquid chromatography method for the determination of the antiviral agent ribavirin in human plasma was developed and validated. The method involved solid-phase extraction on phenyl boronic acid cartridges, a reversed-phase liquid chromatography with a Waters Atlantis dC18 (150 mm x 3.9 mm, 5 microm) column and a mobile phase consisting of 10 mM potassium phosphate buffer (pH 4.0), and ultraviolet detection at 207 nm. This assay proved to be sensitive (lower limit of quantification of 0.05 microg/ml), linear (correlation coefficients >or=0.997), specific (no interference with various potentially co-administrated drugs), reproducible (both intra-day and inter-day coefficients of variation <or=4.3%), and accurate (deviations ranged from -5.6 to 2.2% and from -6.0 to 4.0% for intra-day and inter-day analysis, respectively). The method was applied to therapeutic monitoring of patients undergoing ribavirin treatment for hepatitis C and proved to be robust and reliable. Thus, this method provides a simple, sensitive, precise and reproducible assay for dosing ribavirin that can be readily adaptable to routine use by clinical laboratories with standard equipment. In addition, we evaluated the stability of ribavirin in plasma under various conditions, since no detailed study on thermal stability of ribavirin has been reported so far and discrepant data do exist on ribavirin stability upon conditions that clinical samples commonly experience. Ribavirin was stable in human plasma stored at room temperature for at least 24 h or at -20 degrees C for up to 1 month, after three freeze-thaw cycles, as well as in samples undergoing heat inactivation of infectious viruses for 60 min at 60 degrees C. The drug was also stable in processed samples stored at -20 degrees C for 3 days (as dried extracts) or at 20 degrees C for 4 days (as reconstituted samples).