The canonical members of the human glycoprotein hormone subunit family of cystine knot-forming polypeptides include the common alpha-subunit, and four beta-subunit genes, FSHbeta, LHbeta, TSHbeta, and hCGbeta. Using pairwise sequence analysis of the complete human genome, we have identified two novel glycoprotein hormone subunit-related genes. Based on unique sequence similarity to the alpha- and beta-subunits of glycoprotein hormones, they were named glycoprotein-alpha2 (GPA2) and glycoprotein-beta5 (GPB5), respectively. PCR analysis using a panel of human cDNAs from 14 different tissues demonstrated that GPB5 is similar to other beta-subunits showing restricted tissue expression, mainly in pituitary and brain. In contrast, the GPA2 transcript is found in diverse tissues. Furthermore, immunoreactive GPA2 and GPB5 were detected in the anterior pituitary of mouse and frog, whereas the expression of GPA2 and GPB5 in transfected cells resulted in the secretion of recombinant polypeptides in conditioned medium. After GenBank searches in lower organisms, glycoprotein hormone beta-subunit-related genes were identified from the genome of nematode Caenorhabditis elegans, hookworm Ancylostoma caninum, and Drosophila melanogaster. The evolutionary conservation of these invertebrate homologs can be seen in several key sequence characteristics, and the data suggest that the glycoprotein hormone beta-subunit gene ancestor evolved before the emergence of bilateral metazoa, thus providing a better understanding of the evolution of this group of classic polypeptide hormones and their receptors. Studies of the complete inventory of genes homologous to glycoprotein hormone subunits in the human genome and lower organisms will allow future functional characterization and identification of their respective receptors.