Macrophages accumulate in the vascular wall during conditions such as hypertension and hypercholesterolemia and contribute to vascular remodelling. Here we describe a method for the isolation and subsequent flow cytometric analysis of macrophages from aortas of mice. Cell suspensions were prepared from thoracic aortas of male C57BL/6J mice by a combination of manual disruption, incubation in enzymatic digestion medium, and passage through a 70 μm cell strainer. Flow cytometric analysis of these suspensions revealed a high content of cells with strong light scattering properties (i.e. SSC(hi)) compared with suspensions derived from mouse blood, spleen, thymus or kidney. Unstained aortic cell suspensions also displayed a preponderance of autofluorescence in the B670, V560, V460, B525 and V610 channels of the flow cytometer, suggesting that these channels should be avoided for subsequent flow cytometric analyses, at least for initial gating steps. Thus, aortic preparations were labelled with an APC-Cy7-conjugated antibody against the pan-leukocyte marker, CD45, as well as an APC-conjugated antibody against the macrophage-specific antigen, F4/80, as these fluorochromes emit in channels that displayed relatively low levels of auto-fluorescence in our initial studies (i.e. R780 and R660). Flow cytometric analysis of labelled aortic preparations revealed a distinct population of CD45(+)F4/80(+) cells. Importantly, back-gating on this CD45(+)F4/80(+) cell population showed it to be now virtually devoid of autofluorescence in all remaining open channels, and hence an appropriate foundation for further detailed analysis of macrophage polarization using multiple intra- and extra-cellular markers. Furthermore, we demonstrated that angiotensin II-induced hypertension in C57BL6/J mice, and hypercholesterolemia in apolipoprotein E-deficient mice, each resulted in an approximate doubling of CD45(+)F4/80(+) cells in the aortic wall, highlighting the utility of our new protocol for studying the impact of disease on macrophage accumulation in the vascular wall.