Aims The objectives of this study were to produce Salmonella-specific peptide ligands by phage display biopanning and evaluate their use for magnetic separation (MS). Methods and Results Four-phage display biopanning rounds were performed, and the peptides expressed by the two most Salmonella-specific (on the basis of phage-binding ELISA results) phage clones, MSal020401 and MSal020417, were chemically synthesized and coupled to MyOne tosylactivated Dynabeads (R). Peptide capture capability for whole Salmonella cells from nonenriched broth cultures was quantified by MS+plate counts and MS+Greenlight detection and compared to capture capability of anti-Salmonella (antibody-coated) Dynabeads (R). MS+Greenlight gave a more comprehensive picture of capture capability than MS+plate counts and showed that Peptide MSal020417-coated beads exhibited at least similar, if not better, capture capability to anti-Salmonella Dynabeads (R) (mean capture values of 36 center dot 0 +/- 18 center dot 2 and 31 center dot 2 +/- 20 center dot 1%, respectively, over Salmonella spp. concentration range 3x101-3x106CFUml-1) with cross-reactivity of 1 center dot 9% to three other foodborne pathogens: Escherichia coli, Listeria monocytogenes and Campylobacter jejuni. Conclusions One of the phage display-derived peptide ligands was demonstrated by MS+Greenlight to be a viable antibody alternative for MS of Salmonella spp. Significance and Impact of the Study This study demonstrates an antibody-free approach to Salmonella detection and opens substantial possibilities for more rapid tests for this bacterium.