Fibrin targeting is an attractive strategy for nuclear imaging of thrombosis, atherosclerosis and cancer. Recently, FibPep, an In-111-labeled fibrin-binding peptide, was established as a tracer for fibrin SPECT imaging and was reported to allow sensitive detection of minute thrombi in mice using SPECT. In this study, we developed EPep, a novel In-111-labeled fibrin-binding peptide containing the fibrin-binding domain of the clinically verified EP-2104R peptide, and sought to compare the potential of EPep and FibPep as tracers for fibrin SPECT imaging. In vitro, both EPep and FibPep showed high stability in serum, but were less stable in liver and kidney homogenate assays. Both peptide probes displayed comparable affinities toward human and mouse derived fibrin (K-d approximate to 1 mu M), and similarly to FibPep, EPep showed fast blood clearance, low nontarget uptake and high thrombus uptake (6.8 +/- 1.2% ID g(-1)) in a mouse carotid artery thrombosis model. Furthermore, EPep showed a similar affinity toward rat derived fibrin (K-d approximate to 1 mu M), displayed high thrombus uptake in a rat carotid artery thrombosis model (0.74 +/- 0.39% ID g(-1)), and allowed sensitive detection of thrombosis in rats using SPECT. In contrast, FibPep displayed a significantly lower affinity toward rat derived fibrin (K-d approximate to 14 mu M) and low uptake in rat thrombi (0.06 +/- 0.02% ID g(-1)) and did not allow clear visualization of carotid artery thrombosis in rats using SPECT. These results were confirmed ex vivo by autoradiography, which showed a 7-fold higher ratio of activity in the thrombus over the contralateral carotid artery for EPep in comparison to FibPep. These findings suggest that the FibPep binding fibrin epitope is not fully homologous between humans and rats, and that preclinical rat models of disease should not be employed to gauge the clinical potential of FibPep. In conclusion, both peptides showed approximately similar metabolic stability and affinity toward human and mouse derived fibrin, and displayed high thrombus uptake in a mouse carotid artery thrombosis model. Therefore, both EPep and FibPep are promising fibrin targeted tracers for translation into clinical settings to serve as novel tools for molecular imaging of fibrin.