Previous structure-activity relationship studies of CRF have shown that residues 1-4 were not necessary for receptor binding or transduction, that residues 4-8 were important for activation, and that residues 12-41 were mostly responsible for binding. Finally it was proposed that CRF assumed an alpha-helical structure when interacting with its receptor. By systematic substitution of each residue (except residues 1-4) in ovine CRF (oCRF) by Ala, we have investigated the role played by individual side chains in receptor recognition and activation. Out of 33 analogues (synthesized using SPPS on an MBHA resin, purified by RPHPLC and characterized by amino acid and mass spectral analyses), a significant loss of biological potency (less than 1% potency of native) was observed for 6 analogues ([Ala6], [Ala8], [Ala10], [Ala12], [Ala14], and [Ala38]); 12 analogues had biological potencies ranging from 1% to 60% and ranked as follows: [Ala35] less than [Ala16] less than [Ala9] less than [Ala19] less than [Ala15] less than [Ala13] less than [Ala7] less than [Ala23] less than [Ala11] less than or equal to [Ala21] less than [Ala27] less than or equal to [Ala18]; 8 analogues were found to be equipotent (greater than 60% and less than 150%) ([Ala5], [Ala17], [Ala26], [Ala29], [Ala30], [Ala34], [Ala36], and [Ala37]; and 7 analogues were found to be approximately 2-5 times more potent than native oCRF ([Ala25] = [Ala40] less than or equal to [Ala39] less than or equal to [Ala33] less than [Ala20] less than [Ala22] less than [Ala32], in an in vitro pituitary cell culture assay. In summary, the Ala substitutions which showed the greatest loss of potency (less than 1% of native oCRF) were those replacing hydrophobic residues while those showing the greatest increase in potency were replacing hydrophilic residues. Of the 22 Ala-containing analogues in the C-terminal half of the molecule, 17 analogues have equal or greater potencies than native oCRF. Substitution of Ala in the N-terminal region (residues 5-19) on the other hand is generally detrimental to biological activity. These results suggest that the side chains of residues 5-19 are very important for receptor binding and activation while, in the C-terminal region, the amino acid side chains may be more responsible for structural conservation than for functional expression.