The binding of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC or PAF, platelet-activating factor) to synaptic plasma membranes, microsomal membranes, and other rat cerebral cortex subcellular fractions was studied. Using several PAF-binding antagonists, three distinct sites were identified. Two of them were in intracellular membranes (microsomes) and one in synaptic plasma membranes. Microsomal membranes were prepared after obtaining a 43,500 x g pellet from the postmitochondrial supernatant and subsequent centrifugation at 105,000 x g of the resulting supernatant. Most plasma membrane markers were retained in the 43,500 x g pellet (Sun, G.Y., Huang, H.-M., Kelleher, J.A., Stubbs, E.B., Sun, A. Y. (1988) Neurochem. Int. 12, 69-77). Microsomes were purified by density-gradient centrifugation and marker enzymes showed relatively very low contamination by plasma membrane markers. Myelin and mitochondria were devoid of specific PAF binding. A site displaying the highest PAF-binding affinity reported to date in all cells and membranes (KD = 22.5 +/- 1.7 pM and Bmax 8.75 = fmol/mg protein), was found in the microsomal fraction. There was a second binding site in microsomal fractions (KD = 25.0 +/- 0.8 nM and Bmax = 0.96 pmol/mg protein. Ca2+ decreases PAF affinity for the microsomal binding sites. The third binding site displays relatively low specific PAF binding and is present in synaptosomal plasma membranes. Moreover, displacement curves by a wide variety of PAF antagonists indicated different affinities for each of the binding sites described here. These results indicate that PAF-binding sites are heterogeneous in rat cerebral cortex, and they imply that the microsomal membrane sites may be involved, at least in part, in intracellular events such as gene expression.