The kinetics of phosphorylation of the Sepharose-coupled peptide RRASVA by catalytic subunit of protein kinase A, and diastereomers of this peptide, containing D-amino acids successively in each position, were studied. Coupling of these peptides with the amino and carboxyl termini to CH-and AH-Sepharoses had similar effects on the phosphorylation reaction, increasing the K m and decreasing the V values, respectively. The diastereomeric peptides were also phosphorylated by the enzyme and the rate of this reaction depended on the position of substitution of L-amino acids with their D-analogs. However, this dependence was much less pronounced if compared with stereoselectivity of the enzyme in reactions with these peptides in solution: the K m values for the Sepharose-coupled peptides were almost insensitive to the replacement of L-amino acids with D-analogs and moderate stereoselectivity was revealed in the maximal velocity of the reaction. The Sepharose-coupled peptide containing D-serine was also phosphorylated by protein kinase A while the same peptide in solution did not interact with the enzyme. Consequently, the polymer, enveloping the phosphorylatable peptide, may remarkably influence the recognition of the reaction site, altering both V and K m values.