Random bacteriophage (phage) display peptide libraries have traditionally been used for the selection of clones that bind specific tissues, tumors, and antigens. However, once the targeting peptide is synthetically produced, it often displays a lower affinity than the original phage because of a lack of avidity effects and removal from the virion surface. We hypothesized that multivalent bifunctional phage displaying peptides that target novel molecular biomarkers would facilitate the in vivo imaging of cancer. This study provides proof of principle for the use of phage displaying multiple melanocortin-1 receptor-homing peptides for the pretargeting and subsequent imaging of murine melanomas in vivo.A 2-step melanoma pretargeting-imaging system was developed by first generating and biotinylating phage that displayed up to 5 copies of alpha-melanocyte-stimulating hormone (alpha-MSH) peptide analogs. Second, streptavidin was conjugated to diethylenetriaminepentaacetic acid for the purpose of radiolabeling with (111)In.The specificity of the MSH2.0 phage for the B16-F1 melanoma was demonstrated both in vitro and in vivo. In vitro micropanning assays with phage at inputs of 10(7) and 10(6) transducing units per milliliter resulted in approximately 200- and approximately 1,000-fold-greater recovery of the MSH2.0 phage over the background, respectively. In vivo distribution studies indicated that melanoma uptake values were 2.6 +/- 1.1, 0.6 +/- 0.2, and 1.0 +/- 0.1 (mean +/- SD) percentage injected dose per gram at 0.5, 6, and 24 h after the injection of (111)In-radiolabeled streptavidin ((111)In-SA). The accumulation of radioactivity within the tumor was 1.8 times greater for the biotinylated MSH2.0 phage than for the biotinylated wild-type phage. These data, combined with reduction by 2.4-fold through competition with a nonradiolabeled alpha-MSH peptide analog, indicated the specific targeting of melanoma tumors in vivo. SPECT/CT image analysis of B16-F1 melanoma-bearing mice showed that intravenously injected biotinylated alpha-MSH phage were retained within melanoma tumors at 4 h after injection of (111)In-SA.This study demonstrated the use of multivalent bifunctional phage in a 2-step pretargeting-imaging system.