We have previously shown that after administration of 123[-SP-4 (a synthetic ApoB peptide fragment) to Watanabe heritable hyperlipidemic (WHHL) rabbits that foci of tracer uptake can be identified by external gamma camera imaging which correspond to regions of the aortas found to contain abundant atherosclerotic lesions at postmortem evaluation. Because 99mTc is preferred over 123I for scintigraphic imaging, we prepared a 99mTc-labeled form of the SP-4 peptide, designated 99mTc-P199. To assess the feasibility of detecting atherosclerotic lesions using 99mTc-P199 and to compare the relative uptake of the 99mTc-labeled and radioiodinated peptides by such lesions, an admixture of 99mTc-P199 and 125I-SP-4 was administered to 11 WHHL and 2 normal rabbits. These animals were imaged for up to 3 h and were sacrificed 3–4 h after injection. The extent of aortic lesion involvement and radiotracer uptake were quantitatively compared by planimetric analysis of photographs of the endothelial surface, 99mTc-P199 ex vivo images and 125I-SP-4 autoradiograms of the excised aortas. Pair wise correlation coefficients for planimetric analysis were as follows: photographs versus ex vivo images, r = 0.83, p = 0.003; photographs versus autoradiograms, r = 0.87, p – 0.001; ex vivo images versus autoradiograms, r = 0.83, p = 0.003. 99mTc-P199 in vivo gamma camera images revealed relatively weak focal aortic uptake in 8 of 11 WHHL rabbits manifesting aortic lesions, and focal carotid artery uptake in 4 of 6 WHHL rabbits manifesting carotid lesions. Neither aortic nor carotid foci were visualized in the normal rabbits. We conclude that 99mTc-P199 localizes specifically in atherosclerotic lesions and may be useful for external imaging of atherosclerosis.