A reversed phase system for the HPLC separation of antipyrine and its primary metabolites is described. Based on this system an assay procedure for antipyrine in plasma and saliva was developed with a lowest measurable concentration of 25 ng/ml and precision of +/- 3.6 and +/- 4.5%, respectively. Furthermore, assays for the parent compound, 3-hydroxymethyl-antipyrine, norantipyrine and 4-hydroxy-antipyrine in urine were developed. The lowest measurable concentration for these compounds is about 100 ng/ml except for 3-hydroxymethyl-antipyrine with a lowest measurable concentration of about 200 ng/ml. The precision was established at +/- 3.6 and +/- 5.0% for 3-hydroxymethyl-antipyrine, and antipyrine, respectively, and +/- 7.0 and +/- 3.6% for norantipyrine and 4-hydroxy-antipyrine, respectively. The method was applied to studies on antipyrine metabolism in humans. Following administration of a single dose of 500 mg antipyrine to 5 healthy volunteers, 3.3 +/- 1.2% of the dose was recovered from 48-hour hydrolyzed urine as unchanged drug, 39.7+/- 8.7% as 3-hydroxymethyl-antipyrine, 14.5 +/- 6.8% as norantipyrine and 28.5 +/- 2.2% as 4-hydroxy-antipyrine.