The cerebral metabolism of enantiomers of 5-fluoro-DOPA (5F-DOPA) and 6-fluoro-DOPA (6F-DOPA) was characterized in organotypic cell cultures of fetal rat brain. This system permits the investigation of metabolic processes in brain tissue exclusively, without the effects of peripheral metabolism and transport. Metabolic profiles for each substrate were determined in comparison with those of L-DOPA and D-DOPA. The uptake of DOPA and fluoro-DOPA in aggregating brain cell cultures is strongly preferential for L-enantiomers. Decarboxylation by aromatic L-amino acid decarboxylase is an active step: the major products are dopamine (DA) or 6F-DA and their corresponding products of oxidative deamination, i.e. dihydroxyphenylacetic acid (DOPAC) or 6F-DOPAC, respectively. Decarboxylation products of D-enantiomers occur in lower amounts, and 5F-D-DOPA is not decarboxylated. However, 5F-DOPA is O-methylated to a great extent, and levels of 3-O-methyl-5F-DOPA are higher after incubation with 5F-D-DOPA than with 5F-L-DOPA. These data may serve as a support for more detailed modeling of [18F]F-DOPA metabolism than can be applied to the evaluation of the cerebral biochemistry of the DA system with positron emission tomography in vivo.