The activities of corticotropin-releasing hormone (CRH)-related peptides and several analogs were examined in cells transfected with either CRH1 or CRH2beta receptors, in suppression of heat-induced rat paw edema in pentobarbital-anesthetised animals and in stimulation of release of immunoreactive corticotropin (ir-ACTH) from rat anterior pituitary tissue in vitro. The peptides tested were human/rat (h/r)-CRH, r-urocortin, h-urocortin, white sucker fish or maggy sole urotensin I and some analogs of these peptides substituted with D-amino acids at residues 4 (urocortin), 5 (CRH and urotensin I) and 20 (CRH). In cells transfected with CRH1 receptors, these peptides were similar in potency in stimulation of cAMP accumulation. By contrast, at CRH2beta receptors peptides of the urocortin and urotensin series were more potent than h/r-CRH while [D-Glu20]-h/r-CRH was 6.5-fold less active than h/r-CRH. I.v. administration of h/r-CRH or related peptides 10 min prior to a thermal stimulus produced a significant dose-dependent inhibition of rat paw edema formation. Comparison of the ED50's showed that urocortins ([D-Ser4]-h-urocortin, h-urocortin, [D-Pro4]-r-urocortin, r-urocortin) were approximately 2 to 3 times more active than h/r-CRH, but [D-Glu20]-h/r-CRH was 18.5-fold less active. In the assay for ir-ACTH release, the activity of h/r-CRH and [D-Glu20]-h/r-CRH was similar but [D-Pro5]-h/r-CRH and [D-Pro4]-r-urocortin was less potent than the native peptide. These results provide further evidence that D-amino acid substitution at residue 20 reduces the potency of h/r-CRH at endogenous (anti-edema effect) and transfected (cAMP accumulation) CRH2beta receptors whilst activity at the CRH1 receptor is retained (ACTH-release and cAMP accumulation). On the other hand substitutions at residues 4 or 5 in r-urocortin or h/r-CRH respectively appear to decrease activity at CRH1 but not CRH2beta receptors The modified CRH and urocortin analogs described here may provide clues for the further design of receptor selective ligands.