The study characterizes three distinct populations of activated macrophages (Mφ): classically activated (Ca-Mφ), type II-activated (Mφ-II), and alternatively activated (AA-Mφ). Ca-Mφ are activated by IFN-γ and LPS, while Mφ-II are activated by LPS plus immune complexes, and AA-Mφ by IL-4. The study reveals that Mφ-II more closely resemble Ca-Mφ than AA-Mφ in terms of function and biochemistry. Ca-Mφ and Mφ-II produce high levels of NO and low arginase activity, whereas AA-Mφ express FIZZ1 but not these markers. Ca-Mφ and Mφ-II are efficient antigen-presenting cells (APC), while AA-Mφ are not. Ca-Mφ produce IL-12 and promote Th1 responses, whereas Mφ-II produce IL-10 and promote Th2 responses. Mφ-II express SPHK1 and LIGHT, which may be used to identify them in tissue. These findings suggest that the three populations have distinct biological functions and that the simple classification of non-Ca-Mφ as M2 may be misleading. The study provides a detailed biochemical and functional characterization of these macrophage populations.The study characterizes three distinct populations of activated macrophages (Mφ): classically activated (Ca-Mφ), type II-activated (Mφ-II), and alternatively activated (AA-Mφ). Ca-Mφ are activated by IFN-γ and LPS, while Mφ-II are activated by LPS plus immune complexes, and AA-Mφ by IL-4. The study reveals that Mφ-II more closely resemble Ca-Mφ than AA-Mφ in terms of function and biochemistry. Ca-Mφ and Mφ-II produce high levels of NO and low arginase activity, whereas AA-Mφ express FIZZ1 but not these markers. Ca-Mφ and Mφ-II are efficient antigen-presenting cells (APC), while AA-Mφ are not. Ca-Mφ produce IL-12 and promote Th1 responses, whereas Mφ-II produce IL-10 and promote Th2 responses. Mφ-II express SPHK1 and LIGHT, which may be used to identify them in tissue. These findings suggest that the three populations have distinct biological functions and that the simple classification of non-Ca-Mφ as M2 may be misleading. The study provides a detailed biochemical and functional characterization of these macrophage populations.