A new subfamily member of thyrotropin releasing hormone (TRH) receptor gene, TRHR2, was isolated from rat brain cDNAs. The deduced amino acid sequence of TRHR2 is 51 % identical to that of rat TRH receptor gene which was reported previously. Northern blot analysis with TRHR2 probe revealed brain-specific expression of a 9.5 kb mRNA. In a binding experiment using the TRHR2-expressing COS cells, specific binding of TRH to TRHR2 was observed with Kd value of 9 nM which was equivalent to the Kd value (= 13 nM) of TRH binding to the TRH receptor previously reported. The active metabolite of TRH, histidyl-proline diketopiperazine, or cyclo(His-Pro), showed no specific binding activity. These results suggest that TRHR2 is a novel subtype of TRH receptor. (C) 1998 Academic Press.