A t.l.c.-assay has been developed for the simultaneous determination from the urine of man and animals of three major hydroxylated metabolites of antipyrine (4,4'-dihydroxy-antipyrine, 4-hydroxy-antipyrine, 3-hydroxymethyl-antipyrine). The methodology is also applicable to bile fluid, liver perfusate and liver homogenate. Genuine conjugates are cleaved by acid hydrolysis and free, acid stable metabolites are extracted. Extracts are subjected to t.l.c. and the chromatograms analysed quantitatively by u.v.-reflectance measurements using authentic materials as standards. Calibration curves are linear with a correlation coefficient r greater than 0.990. Recovery for each metabolite is greater than 95%. Reproducibility of the method is good, with variation coefficients in the range of 3-7%, depending on concentration. The sensitivity of the method is sufficient for practical needs. The specificity of the procedure was confirmed using radio-labelled antipyrine. In man, 4-hydroxy-antipyrine is the principal hydroxylation product in this series, accounting for about 35-40% of the dose. 3-Hydroxymethyl-antipyrine makes up for about 13-17% and 4,4'-dihydroxy-antipyrine represents 3-6% of the dose of antipyrine. In the rat, 4-hydroxy-antipyrine accounts for about 15-31%, 3-hydroxymethyl-antipyrine for 22-28% and 4,4'-dihydroxy-antipyrine for up to 11-18% of the dose. Variation of this pattern in different strains is moderate. In both species, the major portion of phase-I metabolites is excreted as conjugates. Part of them appears in a free form.