Identification of novel nuclear receptors based on the highly conserved DNA-binding domain (DBD) has previously depended mainly on low stringency hybridization of cDNA libraries and degenerate PCR. Establishment of the expressed sequence tag (EST) database in recent years has provided an alternative approach for the discovery of novel members of gene families. The rate-limiting step is the conversion of ESTs to full-length cDNA. This article describes the identification of two novel nuclear receptors (hERRβ2 and hERRγ2) related to human estrogen-receptor-related receptor 2 (hERR2) by mining the EST database and retrieving of full-length cDNA via inverse PCR on subdivided primary cDNA library pools. The deduced protein sequences of hERRβ2 and hERRγ2 contain 500 and 458 amino acid (aa) residues respectively. Sequence analysis revealed that hERRβ2 and hERRγ2 respectively share 95% and 77% overall aa sequence identity with hERR2. However, the extra C-terminal domain in hERRβ2 and extra N-terminal domain in hERRγ2 are not present in the closely related hERR2 or mouse ERR2 (mERR2). Extensive sequence verification revealed that hERR2 previously reported as a human gene is actually a rat gene, whereas hERRβ2 is the true human ortholog of hERR2 and mERR2. Tissue distribution studies showed that hERRγ2 was expressed in a broader panel of tissues at a higher level than hERRβ2. hERRβ2 was mapped to cytogenetic locus 14q24.3~-14q31, a region containing multiple loci involved in genetic diseases, including Alzheimer and diabetes. hERRγ2 was mapped to 1q32. Given the high sequence homology between hERRβ2 and mERR2, the two receptors may have similar biological function in vivo.