In the present study, the pharmacological properties of fatty acid amide hydrolase (FAAH) in subcellular fractions of rat brain were investigated using palmitoylethanolamide (PEA) and arachidonyl ethanolamide (anandamide, AEA) as substrates. FAAH hydrolysed [(3)H]PEA in crude homogenates with median K(m) and V(max) values of 2.9 microM and 2.14 nmol.(mg protein)(-1).min(-1), respectively. [(3)H]PEA hydrolysis was inhibited both by non-radioactive AEA (with a K(i) value very similar to the K(m) value for [(3)H]AEA as substrate using the same assay) and by R(-)ibuprofen (mixed-type inhibition K(i) and K'(i) values 88 and 720 microM, respectively). FAAH activity towards both [(3)H]PEA and [(3) myelin = cytosol, but there were no differences between the relative activities towards the two substrates in any of the fractions. [(3)H]PEA hydrolysis in mitochondrial, myelin, microsomal, and synaptosomal fractions was inhibited by oleyl trifluoromethylketone, phenylmethylsulphonyl fluoride, and the R(-)- and S(+)-enantiomers of the nonsteroidal anti-inflammatory drug ibuprofen, with mean IC(50) values in the ranges 0.028-0.041, 0.37-0.52, 67-110, and 130-260 microM, respectively. It is concluded that the pharmacological properties of FAAH in the different subcellular fractions are very similar.