Purpose Atherosclerotic plaque development and progression signifies a complex inflammatory disease mediated by a multitude of proinflammatory leukocyte subsets. Using single photon emission computed tomography (SPECT) coupled with computed tomography (CT), this study tested a new dual-isotope acquisition protocol to assess each radiotracer’s capability to identify plaque phenotype and inflammation levels pertaining to leukocytes expressing leukocyte function-associated antigen-1 (LFA-1) and the leukocyte subset of proinflammatory macrophages expressing somatostatin receptor subtype-2 (SST 2 ). Individual radiotracer uptake was quantified and the presence of corresponding immunohistological cell markers was assessed. Methods Human symptomatic carotid plaque segments were obtained from endarterectomy. Segments were incubated in dual-isotope radiotracers 111 InIn-DOTA-butylamino-NorBIRT ( 111 InIn-Danbirt) and 99m TcTc-N 0–1 4 ,Asp 0 ,Tyr 3 -octreotate ( 99m TcTc-Demotate 2) before scanning with SPECT/CT. Plaque phenotype was classified as pathological intimal thickening, fibrous cap atheroma or fibrocalcific using histology sections based on distinct morphological characteristics. Plaque segments were subsequently immuno-stained with LFA-1 and SST 2 and quantified in terms of positive area fraction and compared against the corresponding SPECT images. Results Focal uptake of co-localising dual-radiotracers identified the heterogeneous distribution of inflamed regions in the plaques which co-localised with positive immuno-stained regions of LFA-1 and SST 2 . 111 InIn-Danbirt and 99m TcTc-Demotate 2 uptake demonstrated a significant positive correlation ( r  = 0.651; p  = 0.001). Fibrous cap atheroma plaque phenotype correlated with the highest 111 InIn-Danbirt and 99m TcTc-Demotate 2 uptake compared with fibrocalcific plaques and pathological intimal thickening phenotypes, in line with the immunohistological analyses. Conclusion A dual-isotope acquisition protocol permits the imaging of multiple leukocyte subsets and the pro-inflammatory macrophages simultaneously in atherosclerotic plaque tissue. 111 InIn-Danbirt may have added value for assessing the total inflammation levels in atherosclerotic plaques in addition to classifying plaque phenotype.