Platelet-Activating Factor (PAF, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is present in nervous tissue and its function is still unknown. We have demonstrated that rat brain is able to synthesize PAF from 1-alkyl-2-acetyl-sn-glycerol and CDP-choline by a "DTT-insensitive" phosphocholine transferase. This represents the last step of the de novo pathway which apparently is the only one existing in the brain for PAF biosynthesis. The enzyme has a microsomal localization, requires Mg++ and is inhibited by Ca++ as reported for phosphocholine transferase utilizing long-chain diradylglycerols as substrates. However, other properties of PAF-synthesizing enzyme (sensitivity to DTT and dependence on pH) are different from those of phosphocholine transferase responsible for the synthesis of diacyl and long-chain alkylacyl glycerophosphocholines. These observations indicate that a specific enzyme for PAF biosynthesis might exist in rat brain.