Radiolabeled alpha-melanocyte stimulating hormone (alpha-MSH) analogs were examined in melanoma-bearing mice to determine the effects of peptide length, structure, and radiometal chelation chemistry on tumor targeting and in vivo biodistribution. The linear alpha-MSH analogs [Nle4,D-Phe7]alpha-MSH (NDPMSH) and [D-Phe7]alpha-MSH(5-10) (DPMSH) were radiolabeled with 99mTc and 188Re via the addition of tetrafluorophenyl mercapto-acetylglycylglycyl-gamma-aminobutyrate (MAG2) or tetrapeptide Ac-Cys-Gly-Cys-Gly (CGCG) chelation moieties. 125I-Tyr2-NDPMSH was obtained by direct iodination of the Tyr2 residue. Tumor uptake of 99mTc-labeled CGCG- and MAG2-NDPMSH analogs at 30 min postinjection were 6.52 +/- 1.11 %ID/g and 4.17 +/- 1.34 %ID/g, respectively, resulting in a significantly higher tumor-to-blood uptake ratio than that of 125I-NDPMSH or a shorter alpha-MSH analog, 99mTc-CGCG-DPMSH. The combination of radiolabeling efficacy and in vivo tumor uptake highlights the potential of 99mTc-CGCG-NDPMSH as a melanoma imaging agent.