Radiation therapy with positively charged particles implies that the Bragg peak be spread out to deliver a homogeneous dose to the tumour. The spread-out Bragg peak (SOBP) has a higher linear energy transfer (LET) than the entrance beam. In addition, there is an LET gradient from proximal to distal SOBP. The aim of this study is to find out whether these small LET variations lead to differences in radiation response. Human melanoma cells (CAL4) were exposed to 65 MeV proton beams produced by the cyclotron Medicyc at five different positions: 2 mm depth corresponding to the entrance, 15, 20, 25 and 26.8 mm depth corresponding to four different positions in the half-modulated SOBP. Survival curves were generated using the in vitro colony method and fitted with the linear-quadratic model. Survival differences were observed at high doses; they were statistically significant at a dose of 8 Gy. With respect to the entrance position (2 mm), the relative biological effectiveness (RBE) at 1% survival was 1.09, 1.12, 1.19 and 1.27 at 15, 20, 25 and 26.8 mm in the SOBP, respectively. Whereas RBE values in the SOBP greater than 1.0 relative to the entrance beam represent a small biological advantage to be added to the well-known physical advantage of high energy proton beams; the RBE gradient along the SOBP would imply that the distal end of the tumour would receive a higher biologically equivalent dose than the proximal end, despite a homogeneous physical dose, especially at the high doses per fraction given in ocular melanomas. Although the increase in effectiveness with depth is mild, it should be kept in mind during eye treatment planning, in case a critical target is present at the extreme end of the SOBP.