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Prostate cancer (PCa) is the second leading cause of cancer death in men and new biomarkers are needed to improve diagnostic and therapeutic approaches in aggressive forms of this disease. CD44v6, a splice variant of the transmembrane glycoprotein CD44, has emerged as a promising biomarker for aggressive PCa and has been shown to be strongly correlated with chemo-and radio-resistant PCa. Here, we hypothesized that aggressive PCa could be radioimaged in vivo by targeting CD44v6 with a CD44v6 specific antibody (Ab). We employed three separate imaging strategies using 67Ga as the radiotracer: a direct-labeled Ab approach, a two-step pre-targeting approach, and a three-step pre-targeting approach. All three strategies were able to image PCa in vivo with varying degrees of success. Tumor uptake in the direct-labeled approach reached 6.61% ID/g but had tumor/liver and tumor/spleen uptake …