We identified two cDNAs encoding new calcitonin receptor-stimulating peptides (CRSPs) in porcine hypothalamus cDNA library by cross-hybridization with the CRSP cDNA, and designated the second and third peptides as CRSP-2 and CRSP-3. The putative amino acid sequences of prepro-CRSP-2 and prepro-CRSP-3 showed higher identity with that of prepro-CRSP-1 than that of prepro-calcitonin gene-related peptide (CGRP), respectively, and these three CRSPs are considered to form a new family in the CGRP superfamily. RT-PCR analysis demonstrated that both CRSP-2 and CRSP-3 gene transcripts were expressed mainly in the central nervous system and thyroid gland. Synthetic CRSP-2 and CRSP-3 stimulated cAMP production very weakly in LLC-PK1 cells compared with CRSP and calcitonin (CT). Furthermore, CRSP-2 and CRSP-3 did not elicit a cAMP elevation at all in the COS-7 cells expressing CT receptor or CT-like receptor with or without one of receptor activity-modifying proteins. These results suggest the presence of still unidentified action mechanisms and functions of the peptides in the CGRP superfamily. (C) 2003 Elsevier Inc. All rights reserved.