AbstractB cells are in analogy with T cells capable of expressing functional IL‐2 receptors. IL‐2R α‐chain (CD25) positive T cells have been studied in detail but not much is known about CD25 positive B cells. The aim of this study was to examine the phenotypic properties of the CD25 expressing B cells collected from different lymphoid organs in mice. Samples were stained for various cell surface markers and analysed using flow cytometry. We found that approximately 49% of B cells in bone marrow, 16% in peritoneal cavity, 2% in spleen and 1% in lymph nodes express CD25. In contrast, CD25 expressing B cells were not found in the blood or in Peyer's patches. Phenotypic characterization showed that CD25+ B cells in spleen, lymph nodes and peritoneal cavity have higher expression of AA4.1, CD5, CD69, CD80, CD86, CD122, CD132, IgA, IgG and IgM on their surface in comparison with CD25− B cells. In contrast, expression of IgD and IA‐IE was lower on CD25+ B cells in spleen and lymph nodes. In bone marrow, the expression of CD5, CD80, CD86, CD122, CD132, IgA, IgD and IgM was lower, while the expression of AA4.1, IgG and IA‐IE was increased on CD25+ B cells compared with CD25− B cells. In conclusion, our results indicate that B cells expressing CD25 are phenotypically distinctly different from those that are CD25 negative. Our findings suggest that CD25+ B cells are more prone to efficient antigen presentation and display a more mature phenotype.