Analogues of the synthetic substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly in which the serine is replaced by other amino acids inhibited the activity of the catalytic subunit of cyclic AMP-dependent protein kinase from beef skeletal muscle (Peak I). All of the analogues were competitive with respect to peptide substrate but apparent Ki values varied depending on the particular amino acid that was substituted for serine. Inhibition was also competitive with respect to mixed histone as determined in experiments utilizing one of the analogues. Acetylation of the terminal amino group of Leu-Arg-Arg-Ala-Ser-Leu-Gly lowered the Km for this substrate from 16 micrometer to 3 micrometer, but a similar modification of the inhibitory analogue Leu-Arg-Arg-Ala-Ala-Leu-Gly resulted in no major change in the Ki value. An amount of inhibitory peptide sufficient to inhibit the cyclic AMP-dependent protein kinase by 90% caused less than 10% inhibition of several cyclic AMP-independent protein kinases indicating a high degree of specificity of inhibition by the peptide analogues. The experiments show that synthetic peptide analogues could be useful in identifying phosphorylation reactions catalyzed by cyclic AMP-dependent protein kinase as distinguished from other protein kinase reactions.