Data on cellular inactivation resulting from mixed irradiation with charged-particle beams of different linear energy transfer (LET) are needed to design a spread-out Bragg peak (SOBP) for heavy-ion radiotherapy. The present study was designed to study the relationship between the physical (LET) and biological (cell killing) properties by using different monoenergetic beams of 3He, 4He and 12C ions (12 and 18.5 MeV/nucleon) and to attempt to apply the experimental data in the design of the SOBP (3 cm width) with a 135 MeV/nucleon carbon beam. Experimental studies of the physical and biological measurements using sequentially combined irradiation were carried out to establish a close relationship between LET and cell inactivation. The results indicated that the dose-cell survival relationship for the combined high- and low-LET beams could be described by a linear-quadratic (LQ) model, in which new coefficients alpha and beta for the combined irradiation were obtained in terms of dose-averaged alpha and square root of beta for the single irradiation with monoenergetic beams. Based on the relationship obtained, the actual SOBP designed for giving a uniform biological effect at 3 cm depth was tested with the 135 MeV/nucleon carbon beam. The results of measurements of both physical (LET) and biological (90% level of cell killing, etc.) properties clearly demonstrated that the SOBP successfully and satisfactorily retained its high dose localization and uniform depth distribution of the biological effect. Based on the application of these results, more useful refinement and development can be expected for the heavy-ion radiotherapy currently under way at the National Institute of Radiological Sciences, Japan.