Platelet-activating factor (l-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) is a potent phospholipid mediator with diverse physiological actions in a wide variety of cells and tissues (1). PAF as well as histamine, prostaglandines and leukotrienes, is thought to play pivotal roles in allergic and inflammatory diseases. Despite the highly hydrophobic structure, PAF binds to a cell-surface, G protein-coupled, seven-transmembrane (TM)-type receptor, which was first cloned in our laboratory (2). So far, PAF receptor homologs in four mammalian species (guinea pig, human, rat, and mouse) have been cloned. Various PAF receptor antagonists have been developed as antiallergic and antiinflammatory drugs, but little is known of ligand binding sites in the cloned PAF receptors. Most G protein-coupled receptor agonists are considered to bind to a hydrophobic core surrounded by seven-TM α-helices with electrostatic and hydrophobic force. We performed alanine-scanning mutagenesis to replace all 23 polar amino acids individually in the putative TM domains of a guinea pig PAF receptor to search for amino acids involved in the recognition of PAF.