Urinary metabolites and 13CO2-production from 13C-octanoic acid were studied in 12 patients (3-19 y.o.) with seizure disorders before and during valproic acid (VPA) therapy without and with L-carnitine supplementation. Only one patient had a decreased plasma level of free carnitine (FC) during VPA. Urinary FC concentrations were positively correlated with serum VPA levels (p<0.01) indicating a dose-related effect of VPA on the tubular reabsorption of FC. Urinary dicarboxylic acid concentrations relative to plasma β-OH-butyrate and urinary hexanoyl- and butyrylglycine concentrations (stable isotope dilution method) increased during VPA. Changes in cumulative excretion of 13CO2 in breath were inconsistent. L-carnitine administration had no significant effect. 11/12 patients excreted octanoylearnitine (OC) in excess of octenoylearnitine during VPA. Quantitation of OC by radioisotopic exchange/HPLC showed urinary concentrations of OC comparable to those found in asymptomatic patients with medium chain acyl-CoA dehydrogenase (MCAD) deficiency (x 0.71; R 0.09-14.6 vs. × 0.38; R 0.10-4.21 mmoles/mole creatinine). Gas chromatographic/mass spectrometric analysis revealed that the predominant saturated 8-carbon acylcarnitine in urine was OC with valproylcarnitine being only a minor metabolite. Conclusion: VPA therapy results in an inhibition of β-oxidation partly at the level of MCAD. L-carnitine has no consistent effect.