Free amino acid enantiomers in adult rat brain extracts were analysed as their N,O‐pentafluoropropionyl isopropyl derivatives by gas chromatography on a capillary column of Chirasil‐L‐Val. A peak X, which exhibited the same retention time as the N,O‐pentafluoropropionyl isopropyl derivative of authentic D‐serine, was detected in the brain extracts. Electron impact and positive chemical ionization mass spectra of the peak X of the brain extracts were identical to those of authentic D‐serine. The concentration of free D‐serine and the ratio of D‐serine/total serine in the brain were estimated to be 0.27 and 0.23, υmol/g of wet weight, respectively. These data provide the first evidence that substantial quantities of free D‐serine are present in mammalian brain tissues.