The recent cloning of a growth hormone secretagogue receptor (GHS-R) from human pituitary gland and brain identified a third G protein-coupled receptor (GPC-R) involved in the control of growth hormone release. The nucleotide sequence of the GHS-R is most closely related to the neurotensin receptor-1 (NT-R1) (35% overall protein identity). Two human GPC-Rs related to both the type 1a GHS-R and NT-Rs were cloned and characterized. Hybridization at low posthybridizational stringency with restriction enzyme-digested human genomic DNA resulted in the identification of a genomic clone encoding a first GHS-R/NT-R family member (GPR38). A cDNA clone was identified encoding a second GHS-R-related gene (GPR39). GPR38 and GPR39 share significant amino acid sequence identity with the GHS-R and NT-Rs 1 and 2. An acidic residue (E124) in TM-3, essential for the binding and activation of the GHS-R by structurally dissimilar GHSs, was conserved in GPR38 and GPR39. GPR38 is encoded by a single gene expressed in thyroid gland, stomach, and bone marrow. GPR39 is encoded by a highly conserved single-copy gene, expressed in brain and other peripheral tissues. Fluorescence in situ hybridization localized the genes for GPR38 and GPR39 to separate chromosomes, distinct from the gene encoding the GHS-R and NT-R type 1. The ligand-binding and functional properties of GPR38 and GPR39 remain to be determined.