We have previously demonstrated preclinical in vivo targeting of prostate stem cell antigen (PSCA) using a humanized anti-PSCA 2B3 monoclonal antibody (mAb). However, humanization resulted in 5-fold loss of apparent affinity relative to the parental mAb (1 nM). In this study, diabodies (scFv dimers of 55 kDa) were generated from 2B3 including variants with different linker lengths as well as back-mutations to original murine residues to improve affinity. Parental 2B3 (p2B3) and back-mutated 2B3 (bm2B3) diabodies (Dbs) with five- or eight-amino acid linkers (p2B3-Db5, p2B3-Db8, bm2B3-Db5 and bm2B3-Db8) were evaluated for binding to PSCA by flow cytometry and affinities were determined by surface plasmon resonance. Back-mutation restored the affinity from 5.4 to 1.9 nM. Stability, evaluated by size exclusion, revealed that diabodies with eight-residue linkers existed as a mixture of dimeric and monomeric species at low concentrations (<or =1 mg/ml). Shortening the linker from eight to five residues improved dimer stability, notably in the bm2B3-Db8 compared with bm2B3-Db5. Both p2B3-Db8 and bm2B3-Db8 were radioiodinated with (124)I and evaluated by serial micro-positron emission tomography imaging in mice bearing LAPC-9 human prostate cancer xenografts. Localization in LAPC-9 xenografts was seen at 4 h, whereas at 20 h most of the activity had cleared from the tumor. Highest tumor-to-background contrast ratios and best images were obtained at 12 h. Although the higher affinity bm2B3-Db8 demonstrated improved tumor retention at later time points (20 h), it did not improve tumor targeting or imaging compared with p2B3-Db8 at 12 h.