Significant upregulation of the integrin alpha(v)beta(6) has been described as a prognostic indicator in several cancers, making it an attractive target for tumor imaging. This study compares variants of a PEGylated alpha(v)beta(6)-targeting peptide, bearing either an [(18)F]fluorobenzoyl prosthetic group ([(18)F]FBA-PEG-A20FMDV2) or different [(64)Cu]copper chelators (DOTA-PEG-A20FMDV2, CB-TE2A-PEG-A20FMDV2). The compounds were evaluated in vitro by enzyme-linked immunosorbent assay (against the integrin alpha(v)beta(6) and the closely related integrin alpha(v)beta(3)) and by cell labeling (alpha(v)beta(6)-positive DX3purobeta6/alpha(v)beta(6)-negative DX3puro) and in vivo using micro-positron emission tomography in a mouse model bearing paired DX3purobeta6/Dx3puro xenografts. In vitro, all three compounds showed excellent alpha(v)beta(6)-specific binding (50% inhibitory concentration [IC(50)](alpha(v)beta(6)) = 3 to 6 nmol/L; IC(50)(alpha(v)beta(3)) > 10 micromol/L). In vivo, they displayed comparable, preferential uptake for the alpha(v)beta(6)-expressing xenograft over the alpha(v)beta(6)-negative control (> 4:1 ratio at 4 hours postinjection). Whereas [(64)Cu]Cu-DOTA-PEG-A20FMDV2 resulted in increased levels of radioactivity in the liver, [(64)Cu]Cu-CB-TE2A-PEG-A20FMDV2 did not. Significantly, both (64)Cu-labeled tracers showed unexpectedly high and persistent levels of radioactivity in the kidneys (> 40% injected dose/g at 4 and 12 hours postinjection). The findings underscore the potential influence of the prosthetic group on targeted in vivo imaging of clinically relevant markers such as alpha(v)beta(6). Despite identical targeting peptide moiety and largely equal in vitro behavior, both (64)Cu-labeled tracers displayed inferior pharmacokinetics, making them in their present form less suitable candidates than the (18)F-labeled tracer for in vivo imaging of alpha(v)beta(6).