Purpose: Human tumors are heterogeneous in various biological features including radiosensitivities. We here studied significance of tumor heterogeneity in RBE of carbon ion therapy by using artificially mixed and transplanted experimental tumors in syngeneic mice. Materials and Methods: Three fibrosarcomas (#6107, #8697 and #9037) with similar growth rates were transplanted into C3H male mice. Cell suspensions prepared from 2 fibrosarcomas were mixed at the time of transplantation into hind legs of mice. Grew to 7-8 mm in diameter, tumors were locally irradiated with single doses of 74 keV/μm carbon ions or Cs-137 γ rays. Tumor diameters were measured after irradiation by calipers and tumor growth delay (TGD) time was calculated from growth curves. Five mice were used for each dose group. RBE values of carbon ions relative to γ rays were calculated by comparing isoeffect doses on dose-response curves. Results: Mixture ratios varied from 0:100, 10:90, 50:50, 90:10 and 100:0. Three combinations, i.e., #6107+#8697, #6107+#9037 and #8697+#9037,of the various mixture ratios provided 12 tumors with different heterogeneity. An isoeffect dose to produce TDG times of 15 days was used to calculate an BRE value on a dose response curve. Plotting all 12 RBE values on either γ-ray doses or carbon ion dose, the followings were obtained (1) Isoeffect doses distributed between 25 and 44 Gy for γ rays while between 6.5 and 25 Gy for carbon ions. (2) RBE values depended on carbon-ion sensitivities, and were larger for sensitive tumors than resistant tumors. RBE was independent of γ-ray sensitivities. (3) Any combinations of carbon-ion sensitive tumors (#6107 and #8697) showed RBE values either similar to or larger than parent tumors. (4) Any combinations of carbon-ion sensitive and resistant tumors showed RBE values larger than that of the resistant parent. Namely, combinations of carbon-ion sensitive (#8697) and resistant (#9037) tumors showed RBE values similar to the RBE of parental #8697 tumors while combinations of carbon-ion sensitive (#6107) and resistant (#9037) tumors showed RBE values between those of two parental tumors.Conclusions: RBE of carbon ions depended on carbon-ion sensitivities but not γ-ray sensitivities of tumors. It was carbon-ion sensitive tumors but not resistant tumors that determine an RBE of a heterogeneous tumor.