The present study aimed to discriminate different subsets of cultured dendritic cells (DCs) to evaluate their immunological characteristics. DCs offer an important foundation for immunological studies, and mouse bone marrow (BM) cells cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) have been used extensively to generate CD11c(+)/major histocompatibility complex II+ BM-derived DCs (BMDCs). Immature DCs are considered to have strong migration and phagocytic antigen-capturing abilities, whereas mature DCs are thought to activate naive T cells and express high levels of costimulatory cytokines and adhesion molecules. In most culture systems, non-adherent cells are collected as mature and qualified DCs, and the remaining adherent cells are discarded. The output from GM-CSF cultures comprises mostly adherent cells, and only a small portion of them is non-adherent. This situation has resulted in ambiguities in the attempts to understand results from the use of cultured DCs. In the present study, DCs were divided into three subsets: i) Non-adherent cells; ii) adherent cells and iii) mixed cells. The heterogeneous features of cultured DCs were identified by evaluating the maturation status, cytokine secretion and the ability to activate allogeneic T cells according to different subsets. Results from the study demonstrated that BMDC culture systems were a heterogeneous group of cells comprising non-adherent cells, adherent cells, mixed cells and firmly adherent cells. Non-adherent cells may be used in future studies that require relatively mature DCs such as anticancer immunity. Adherent cells may be used to induce tolerance DCs, whereas mixed cells may potentiate either tolerogenicity or pro-tumorigenic responses. Firmly adherent cells were considered to have macrophage-like properties. The findings may aid in immunological studies that use cultured DCs and may lead to more precise DC research.