A large family of TRH-like peptides in the limbic region of rat brain including pGlu-Glu-Pro-NH(2) (EEP), pGlu-Val-Pro-NH(2) (Val(2)-TRH), Leu(2)-TRH, Phe(2)-TRH and Tyr(2)-TRH has recently been discovered. TRH (pGlu-His-Pro-NH(2)) has antidepressant, neuroprotective, analeptic, anticonvulsant, antiamnesic and euphoric properties, and other TRH-like peptides such as EEP exert several of these effects. A new TRH receptor (TRHR2) has been reported which is highly expressed in regions of rat brain that regulate attention and learning, arousal, sleep and processing of sensory information. The TRHR1 predominates in limbic structures involved in regulation of mood and in pituitary. This study examined the possibility that some of the newly discovered TRH-like peptides bind with high affinity to TRHR2, and that this receptor acts as the transducer for some of the CNS effects of this new class of neuropeptides. EEP, Val(2)-TRH and Leu(2)-TRH were analeptics, like TRH, but Phe(2)-TRH and Tyr(2)-TRH were not. The affinity and efficacy of TRH-like peptides for TRHR1 and TRHR2 were measured in HEK293 cells stably expressing these receptors. The IC(50) values of TRH-like peptides for displacement of [3H]TRH from TRHR2 were TRH<<<(Leu(2)-, Phe(2)-TRH)<(Gln(2)-, Ser(2)-TRH)<<(Val(2)-, Tyr(2)-, Arg(2)-, Thr(2)-, and Glu(2)-TRH). The IC(50) for Leu(2)-TRH was about 100 times that for TRH. When tested at the calculated IC(50) values, TRH-like peptides stimulated calcium responses in cells expressing TRHR1 and TRHR2, indicating that the peptides act as weak agonists at both receptors. These results indicate that TRHR1 and TRHR2 do not mediate the behavioral effects of TRH-like peptides.