Biopanning of tumor cells was used in order to identify matrix metalloproteinase 9 (MMP-9) targeting peptides. The tumor cell targeting peptide (TCTP-1) and two modified versions thereof were evaluated as imaging agents for positron emission tomography (PET) using a rat melanoma xenograft model. For the PET imaging purposes, the 3 peptides were 1,4,7,10-tetraazacyclo-dodecane-N',N '',N''',N ''''-tetraacetic acid (DOTA) conjugated and labeled with Gallium-68 (Ga-68) and preliminarily evaluated: (1) cyclic Ga-68-DOTA-TCTP-1 with cystine bridge, (2) cyclic Ga-68-DOTA-lactam-TCTP-1 with a lactam bridge, and (3) linear Ga-68-DOTA-lin-TCTP-1. The whole-body distribution kinetics and tumor targeting of the intravenously administered Ga-68-DOTA-peptides were evaluated in vivo by PET and ex vivo by measuring the radioactivity of excised tissues. In addition, the in vivo stability of the radiolabeled peptides in rat plasma, tumor tissue, and urine was studied. All Ga-68-DOTA-peptides were cleared via the liver and kidneys, and approximately 44% of injected radioactivity was excreted in urine during 120 min after injection. Ex vivo biodistribution studies showed a tumor-to-muscle ratio of 5.5 +/- 1.3 (mean +/- SD) for Ga-68-DOTA-TCTP-1, 3.2 +/- 0.2 for Ga-68-DOTA-lactam-TCTP-1, and 3.2 +/- 0.6 for Ga-68-DOTA-lin-TCTP-1 at 120 min after injection. The Ga-68-DOTA-lactam-TCTP-1 peptide appeared to be the most stable in vivo. The fraction of intact Ga-68-DOTA-lactam-TCTP-1 in tumor was 59 +/- 4.2% at 120 min after injection. The stability was moderate for Ga-68-DOTA-TCTP-1 and poor for Ga-68-DOTA-lin-TCTP-1. The possibility of imaging tumors that overexpress MMP-9, such as melanoma, by using radiolabeled TCTP peptides in PET imaging makes these peptides highly attractive for diagnostic and therapeutic applications. However, further modifications to improve the stability and affinity of the peptides are needed.