Peptide 1, Leu-Arg-Arg-Ala-Ser-Leu-Gly, is an excellent substrate for cAMP-dependent protein kinase. While the importance of both arginines for effective enzyme-substrate interactions has been shown, it has not been known whether the kinase will catalyze phosphorylation of substrates which contain other than peptide bonds. We report that analogs of peptide 1 which contain depsi linkages replacing selected amide bonds are good protein kinase substrates. Therefore, with the possible exception of the serine amide proton, no peptide 1 amide hydrogens are involved in peptide-peptide or peptide-enzyme hydrogen bonding crucial to defining the high substrate activity of this peptide. It is thus unlikely that peptide 1 is bound by the protein kinase while in an alpha-helical or a beta-turn structure. Three peptides were found to be very poor substrates for protein kinase, those containing N-methyl amino acids in place of Ser5 or Leu6 and a peptide containing Pro in place of Leu6. These peptides are poor substrates for the enzyme possibly because they are unable to adopt a conformation necessary for catalysis of phosphoryl group transfer to occur or due to steric effects in the enzymatic active site.