Introduction Tumour-associated urokinase plasminogen activator (uPA) is a critical marker of invasion and metastasis, and it is recognised as having strong prognostic relevance as well as being a therapeutic target. The specific uPA inhibitor plasminogen activator inhibitor type-2 (PAI-2, SerpinB2) specifically targets cell bound uPA and is internalised. Furthermore, preclinical studies have established the "proof-of-principle" of uPA-targeting by PAI-2-cytotoxin conjugates in human carcinoma models. However, these studies also suggest that PAI-2 is rapidly cleared via the renal system with low total dose reaching the tumour. In this study, a comparative single photon emission computed tomography (SPECT) and biodistribution (BD) analysis of different forms of PAI-2 labelled with the radioisotopes iodine-123 ( 123 I) and technetium-99m ( 99m Tc) was undertaken. Methods The pharmacokinetic (PK) properties and BD of wild-type, ΔCD-loop and PEGylated ΔCD-loop PAI-2 labelled with the commonly used diagnostic SPECT radioisotopes 99m Tc or 123 I were compared in mouse models of human prostate carcinoma. Whole body SPECT imaging was also performed. Results Both wild-type and the shorter but active ΔCD-loop form of PAI-2 123 I-labelled indirectly via conjugation to free amine groups (termed 123 I-Bn-PAI-2) exhibited low tumour uptake, rapid excretion and similar PK profiles. Preliminary studies with a short branched-chain PEGylated 123 I-Bn-PAI-2 ΔCD-loop indicated an increase in blood retention time and tumour uptake. All 123 I-Bn-labelled radiotracers were largely excreted through the kidneys. By comparison, both wild-type 123 I-PAI-2 (labelled directly via tyrosine residues) and 99m Tc-PAI-2 displayed different PK/BD patterns compared to 123 I-Bn-PAI-2, suggesting greater liver based catabolism and thus slower elimination. SPECT imaging mimicked the BD results of all radiotracers. Conclusion The different labelling methods gave distinct PAI-2 BD and tumour uptake profiles, with radioiodination resulting in the best non-tumour organ clearance profiles. Preliminary analyses with short branched-chain PEGylated 123 I-Bn-PAI-2 ΔCD-loop suggest that further investigations with other PEGylation reagents are required to optimise this approach for tumour imaging. These findings impact on the use of PAI-2 for drug delivery and/or diagnostic development.