In vertebrates, recognition of odorous compounds is based on a large repertoire of receptor subtypes encoded by a multigene family. Towards an understanding of the phylogenetic origin of the vertebrate olfactory receptor family, attempts have been made to identify related receptor genes in the river lampreys (Lampetra fluviatilis), which are descendants of the earliest craniates and living representatives of the most ancient vertebrates. Employing molecular cloning approaches led to the discovery of four genes encoding heptahelical receptors, which share only a rather low overall sequence identity but several of the characteristic structural hallmarks with vertebrate olfactory receptors. Furthermore, in situ hybridization studies demonstrated that the identified genes are expressed in chemosensory cells of the singular lamprey olfactory organ. Molecular phylogenetic analysis confirmed a close relationship of the lamprey receptors to vertebrate olfactory receptors and in addition demonstrated that olfactory genes of the agnathostomes diverged from the gnathostome receptor genes before those split into class I and class II receptors. The data indicate that the lamprey receptors represent the most ancient family of the hitherto identified vertebrate olfactory receptors. (C) 1999 Elsevier Science B.V. All rights reserved.