G protein-coupled receptors (GPCRs) are integral membrane proteins that mediate signals to the interior of the cell via activation of heterotrimeric G proteins, which subsequently interact with and activate various effector proteins, ultimately resulting in a physiological response. GPCRs are involved in a number of behaviours, and have been implicated in such processes as drug addiction and neuropsychiatric diseases. To further our understanding of the molecular mechanisms involved beyond the known GPCR-endogenous ligand systems we endeavored to discover novel GPCRs, in particular opioid and opioid related receptors, potentially involved in these processes. In order to achieve this we applied two distinct homology cloning strategies to identify the DNA sequences encoding novel GPCRs. Firstly, PCR amplification of human genomic DNA and cDNA using degenerate oligonucleotides derived from the transmembrane domains (TMs) of the opioid and the related somatostatin receptors, and secondly, querying publicly available sequence databases using the amino add sequence of known GPCRs. We have discovered a novel family comprised of two receptors, named 'GPR7' and 'GPR8', and a third receptor, named 'GPR14', that are most closely related to the opioid receptors. Our experiments also led to the cloning and successful ligand assignment of a novel P2 receptor subtype, named P2Y4, that selectively binds uridine nucleotides. We also report the discovery of three P2Y4 related receptors, ' GPR20, GPR34', and 'GPR35'. We also successfully identified an additional novel family comprised of three receptors, 'GPR3, GPR6 ', and 'GPR12', most closely related to the cannabinoid receptors. We also identified a novel GPCR, named GPR26, which is activated by high LPA concentrations (EC50 = 0.5 [mu]M). The other receptors we isolated, ' GPR1, GPR30, GPR32, GPR33' are all related to chemoattractant receptors, and we have identified two related GPCR pseudogenes, 4 GPR32 and 4 GPR33. The endogenous ligands for most of these receptors have not been assigned, hence they are referred to as orphan receptors, and are useful targets for endogenous ligand discovery. Examination of the mRNA tissue expression patterns by Northern blot and 'in situ' hybridization revealed that most are abundantly and discretely expressed in the brain, suggesting a unique role for each of these receptors in the central nervous system (CNS). Fluorescence in situ hybridization (FISH) was performed to assign each gene to a human chromosome. The cloning and identification of these novel GPCRs provide a unique opportunity for ligand discovery, and is the critical first step in the identification of many novel endogenous ligand-receptor systems.