Summary To assess the achievement of uniformity of radiobiological effectiveness at different depths in the proton spread-out Bragg peak (SOBP), Chinese hamster ovary (CHO) cells were exposed to 65-MeV modulated proton beams at the Research Center for Nuclear Physics (RCNP) of Osaka University. We selected four different irradiation positions: 2 mm depth, corresponding to the entrance, and 10, 18 and 23 mm depths, corresponding to different positions in the SOBP. Cell survival curves were generated with the in vitro colony formation method and fitted to the linear-quadratic model. With 137CS gamma-rays as the reference irradiation, the relative biological effectiveness (RBE) values for a surviving fraction (SF) level of 0.1 are 1.05, 1.10, 1.12 and 1.19 for depths of 2, 10, 18 and 23 mm respectively. A significant difference was found between the survival curves at 10 and 23 mm (P < 0.05), but not between 18 and 10 mm or between 18 and 23 mm. There was a significant dependence of RBE on depths in modulated proton beams at the 0.1 surviving fraction level (P< 0.05). Moreover, the rise of RBEs significantly depended on increasing SF level or decreased approximately in correspondence with irradiation dose (P = 0.0001). To maintain uniformity of radiobiological effectiveness for the target volume, careful attention should be paid to the influence of depth of beam and irradiation dose.Keywords: proton; spread-out Bragg peak; relative biological effectiveness; Chinese hamster ovary cellThe depth-dose curve for the single-energy proton beam has limited applications in clinical radiation therapy owing to the excessively narrow high-dose region. This region, also known as the Bragg peak, can be modulated by appropriate selection of a distribution of proton energies to produce a spread-out Bragg peak (SOBP) or a uniform region of full dose at the depth of interest. Dose uniformity across a target volume can be achieved with multiple X-ray beams. However, each X-ray beam features a greater dose in the entrance region than a corresponding proton beam, has a dose gradient across the target volume and delivers an undesirable dose to normal tissues distal to the target. Proton beams have none of these undesirable characteristics (Suit and Urie, 1992; Munzenrider and Crowell, 1994;Raju, 1996).Although SOBP produces an excellent physical dose distribution, there is a variation of linear energy transfer (LET) values at different depths in the SOBP, known as the LET gradient, from proximal to distal SOBP. The proton is a lower LET particle than other heavy-charged particles; for example, the mean LET values of the 65-MeV modulated proton beams (SOBP) are always less than 7 keV .m-' (Courdi et al, 1994).The achievement of uniformity of radiobiological effectiveness for target volumes is always a matter of concern. In fact, there is no uniformity of LET within target volumes. One study has suggested that DNA double-strand breaks, potentially lethal damage and sublethal damage, depend on LET and are closely