Macrophages represent myeloid lineage cells, resident in many if not every tissue throughout the entire body. They patrol within tissues and participate in maintaining homeostasis. In a case of acute infection they easily differentiate in proinflammatory macrophages (M1) necessary to efficiently confront invading pathogen, secreting different cytokines, chemokines and other inflammatory mediators. Upon activation macrophages express number of genes de novo, including gene for the iNOS. In our study we used intracellular staining with monoclonal antibody for iNOS to evaluate classical differentiation into M1 type after cultivation of peritoneal mouse macrophages in medium with LPS and IFN–γ. Both compounds induced iNOS expression after 24h that maintained high after 72 hours. Due to high plasticity, macrophages can differentiate in opposite M2 type with a role in sustaining overwhelming inflammation and support resolution. This alternative differentiation also results in a gene expression change and could be monitored by intracellular Arg1 detection. In our study IL–4 supplementation induced de novo Arg1 expression after 24 hours that was upregulated after 72 hours. This study confirmed ability of isolated mouse peritoneal macrophages to differentiate into M1 and M2 type by modifying culture settings that can be analyzed by flow cytometer, detecting intracellular iNOS and Arg1 expression together with specific surface macrophage markers.