In order to determine which amino acid side chains of ovine corticotropin releasing factor (oCRF) are most sensitive to alterations with respect to receptor binding and activation, we synthesized a single-point replacement set by replacing each residue by a similar, preferably proteinogenic amino acid, maintaining a minimal change of character at each position (Ser by Thr, Gln by Asn, Glu by Asp, Arg by Lys, and vice versa, Pro by N-MeAla, Ile by Leu, Leu by Nle, Phe by Trp, His by Ala, Val by Leu, Met by Nle, Ala by Leu). In general, any loss in the biological potency by a single-point substitution in oCRF parallels a decrease in receptor binding, indicating that, in contrast to previous suggestions, there is no specific side chain in the peptide that is more responsible for receptor activation than for receptor binding. In addition to Arg(16), Ala(31), and Arg(35), amino acid residues in the N-terminal sequence (5-14) were found to be sensitive to alteration, demonstrating their particular importance for the receptor interaction of CRF agonists. Most of the analogs tested exhibited agonistic potencies in an in vitro pituitary cell culture assay at a concentration of 0.3 nM, and all analogs showed full agonistic potency at 1 microM. In contrast to the results of an alanine replacement study, the strongest decrease in receptor binding and biological potency was observed for analogs with substitutions of hydrophilic amino acids Ser(7), Arg(16), Glu(17), or Asn(34). In the case of Ser(7) and Arg(16), side chain specific interactions with the receptor may be required for high affinity. Alanine replacements at positions 17 or 34 resulted in analogs that were as potent as oCRF, while replacement of Glu(17) by Asp or Asn(34) by Gln caused a dramatic loss in potency, thereby suggesting an important effect at sterically or conformationally sensitive positions. In contrast to corresponding alanine analogs which exhibited a significant loss in biological potency, slight alterations of lipophilic side chains at positions 6, 12, or 38 did not cause a significant reduction of receptor binding and activation, indicating that it is not specific side chains but rather lipophilicity which is essential at these positions. Indeed, replacement of Phe(12) by Trp provides an agonist with significantly increased receptor binding and biological potency.