BACKGROUND: While chimeric antigen receptor (CAR) T-cells are promising, there is a rapidly growing interest in developing other CAR-expressing immune cells. However, to date, no reported studies evaluated these cells side-by-side in immune-competent glioma models. METHODS: We developed a novel C57BL/6-background transgenic mouse strain with all hematopoietic cells carrying the anti-epidermal growth factor receptor (EGFR)vIII-CAR downstream of a Lox-Stop-Lox cassette in the Rosa26 locus. Crossing with mice transgenic for Vav-Cre allowed the expression of anti-EGFRvIII CAR in all hematopoietic cells. In particular, we evaluated CAR-T, CAR-NKT, CAR-NK-cells, and CAR-macrophages in a syngeneic mouse SB28EGFRVIII glioma model. RESULTS: CAR-NK and CAR-NKT-cells demonstrated anti-tumor effects comparable to CAR-T cells in vitro. A single intratumoral administration of CAR-T and CAR-NKT cells in combination mediated superior therapeutic efficacy compared to CAR-T cells or CAR-NKT-cells alone. A single intravenous infusion of CAR-NK cells following lymphodepletion failed to mediate significant anti-glioma effects. Additionally, intratumoral injection of CAR-NK cells did not confer therapeutic benefit. Contrary to previous reports using human macrophages, CAR-macrophages did not demonstrate enhanced antigen-presentation activity against glioma cells compared to non-CAR macrophages. Intratumorally administered CAR-macrophages failed to demonstrate local persistence or anti-tumor effects in vivo. CONCLUSIONS: These data provide a valuable basis as to which immune cells can mediate effective anti-glioma response in an immuno-competent glioma environment. Our data also suggest that a combination of CAR-T and CAR-NKT-cells may represent a promising therapeutic strategy.