Echovirus 22 (EV22) is a picornavirus forming a distinct molecular cluster together with echovirus 23. EV22 has an Arg-Gly-Asp (RGD) peptide motif in its capsid protein VP1; similar motifs are known to mediate many cell-cell and microbe-host interactions. To identify peptide sequences that specifically bind to EV22 and potentially play a role in receptor recognition, we have used here peptide libraries displayed in filamentous phage. We isolated an EV22-binding motif CLRSG(R/F)GC. The synthetic CLRSGRGC peptide was able to inhibit EV22 infection. The infection was also inhibited by an RGD-containing peptide representing the C terminus of the EV22 capsid protein VP1 and CWDDGWLC (an RGD-binding peptide; Pasqualini, R., Koivunen, E., and Ruoslahti, E. (1995) J. Cell Biol. 130, 1189-1196). As the EV22-recognizing sequence LRSG is found in the integrin beta1 chain and the entire LRSGRG hexapeptide occurs in the matrix metalloproteinase 9 (MMP-9), we carried out blocking experiments with anti-integrin and anti-MMP-9 antibodies. EV22 infection could be blocked in cell cultures with anti-alphav, -beta1, and, to a lesser extent, with anti-MMP-9 antibodies. These results imply that EV22 recognizes preferentially alphavbeta1-integrin as a cellular receptor and MMP-9 may also play a role in the cell-surface interactions of the virus.