The recently reported CXCR4 antagonist 3 (Ac-Arg-Ala-[DCys-Arg-2Nal-His-Pen]-CO2H) was investigated as a molecular scaffold for a CXCR4-targeted positron emission tomography (PET) tracer. Toward this end, 3 was functionalized with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 1,4,7-triazacyclononanetriacetic acid (NOTA). On the basis of convincing affinity data, both tracers, [68Ga]NOTA analogue ([68Ga]-5) and [68Ga]DOTA analogue ([68Ga]-4), were evaluated for PET imaging in "in vivo" models of CHO-hCXCR4 and Daudi lymphoma cells. PET imaging and biodistribution studies revealed higher CXCR4-specific tumor uptake and high tumor/background ratios for the [68Ga]NOTA analogue ([68Ga]-5) than for the [68Ga]DOTA analogue ([68Ga]-4) in both in vivo models. Moreover, [68Ga]-4 and [68Ga]-5 displayed rapid clearance and very low levels of accumulation in all nontarget tissues but the kidney. Although the high tumor/background ratios observed in the mouse xenograft model could partially derive from the hCXCR4 selectivity of [68Ga]-5, our results encourage its translation into a clinical context as a novel peptide-based tracer for imaging of CXCR4-overexpressing tumors.