Melanoma is a malignant tumor derived from epidermal melanocytes, and it is known for its aggressiveness, therapeutic resistance, and predisposition for late metastasis. Very late antigen-4 (VLA-4; also called integrin a4β1) is a transmembrane noncovalent heterodimer overexpressed in melanoma tumors that plays an important role in tumor growth, angiogenesis, and metastasis by promoting adhesion and migration of cancer cells. In this study, we evaluated 2 conjugates of a high-affinity VLA-4 peptidomimetic ligand, LLP2A, for PET/CT imaging in a subcutaneous and metastatic melanoma tumor. Methods: LLP2A was conjugated to 1,4,8,11-tetraazacyclotetradecane-1-(methane phosphonic acid)-8-(methane carboxylic acid) (CB-TE1A1P) and 2-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)pentanedioic acid (NODAGA) chelators for 68Ga and 64Cu labeling. The conjugates were synthesized by solid-phase peptide synthesis, purified by reversed-phase high-performance liquid chromatography, and verified by liquid chromatography mass pectrometry. Saturation and competitive binding assays with B16F10 melanoma cells determined the affinity of the compounds for VLA-4. The biodistributions of the LLP2A conjugates were evaluated in murine B16F10 subcutaneous tumor-bearing C57BL/6 mice. Melanoma metastasis was induced by intracardiac injection of B16F10 cells. PET/CT imaging was performed at 2, 4, and 24 h after injection for the 64Cu tracers and 1 h after injection for the 68Ga tracer. Results: 64Cu-labeled CBTE1A1P- PEG4-LLP2A and NODAGA-PEG4-LLP2A showed high affinity to VLA-4, with a comparable dissociation constant (0.28 vs. 0.23 nM) and receptor concentration (296 vs. 243 fmol/mg). The tumor uptake at 2 h after injection was comparable for the 2 probes, but 64Cu-CB-TE1A1P-PEG4-LLP2A trended toward higher uptake than 64Cu-NODAGA-PEG4-LLP2A (16.9 ± 2.2 vs. 13.4 ± 1.7 percentage injected dose per gram, P 5 0.07). Tumor-to-muscle and tumorto- blood ratios from biodistribution and PET/CT images were significantly higher for 64Cu-CB-TE1A1P-PEG4LLP2A than 64Cu- NODAGA-PEG4-LLP2A (all P values , 0.05). PET/CT imaging of metastatic melanoma with 68Ga-NODAGA-PEG4-LLP2A and 64Cu- NODAGA-PEG4-LLP2A showed high uptake of the probes at the site of metastasis, correlating with the bioluminescence imaging of the tumor. Conclusion: These data demonstrate that 64Cu-labeled CBTE1A1P/ NODAGA LLP2A conjugates and 68Ga-labeled NODAGALLP2A are excellent imaging agents for melanoma and potentially other VLA-4-positive tumors. 64Cu-CB-TE1A1P-PEG4-LLP2A had the most optimal tumor-to-nontarget tissue ratios for translation into humans as a PET imaging agent for melanoma.