Protein kinase substrate phage (PKS phage) was constructed by fusing the substrate recognition consensus sequence of CAMP-dependent protein kinase (cAPK) with bacteriophage minor coat protein g3p and by displaying it on the surface of filamentous bacteriophage fd. Phosphorylationin vitro by cAPK showed a unique labelled band of approximately 60 ku, which was consistent with the molecular weight of the PKS-g3p fusion protein. Some weakly phosphorylated bands for both PKS phage and wild-type phage were also observed. Phage display random 15-rner peptide library phosphorylated by cAPK was selected with ferric (Fe(3+)) chelation affinity resin. After 4 rounds of screening, phage clones were picked out to determine the displayed peptide sequences by DNA sequencing. The results showed that 5 of 14 sequenced phages displayed the cAPK recognition sequence motif (R)RXS/T. Theirin vitro phosphorylation analyses revealed the specific labelled bands corresponding to the positive PKS phages with and without the typical (R)RXS/T sequence motif. It suggested that the new method of using ferric (Fe(3+)) chelation affinity chromamraphy to identify the substrate specificity of protein kinase from random peptide library was feasible.