Cerenkov luminescence imaging (CLI) can provide high-resolution images of (18)F-FDG-avid tumors but requires prolonged acquisition times because of low photon sensitivity. In this study, we proposed a new modality, termed beta-radioluminescence imaging (beta-RLI), which incorporates a scintillator with a gamma-rejection strategy for imaging beta particles. We performed a comparative evaluation of beta-RLI with CLI in both in vitro and in vivo systems. METHODS: Using in vitro phantoms, we characterized the photon sensitivity and resolution of CLI and beta-RLI. We also conducted a series of in vivo experiments with xenograft mouse models using both amelanotic (A375, UMSCC1-Luc) and melanotic (B16F10-Luc) cell lines. The B16F10 and UMSCC1 cell lines were transfected with the luciferase gene (Luc). CLI was acquired over 300 s, and beta-RLI was acquired using two 10-s acquisitions. We correlated (18)F -: FDG activities, as assessed by PET, with tumor radiances for both beta-RLI and CLI. We also compared tumor signal-to-background ratios (SBRs) between these modalities for amelanotic and melanotic tumors. RESULTS: For in vitro experiments, the photon sensitivity for beta-RLI was 560-fold greater than that for CLI. However, the spatial resolution for beta-RLI (4.4 mm) was inferior to that of CLI (1.0 mm). For in vivo experiments, correlations between (18)F-FDG activity and tumor radiance were 0.52 (P < 0.01) for beta-RLI, 0.81 (P = 0.01) for amelanotic lesions with CLI, and -0.08 (negative contrast; P = 0.80) for melanotic lesions with CLI. Nine of 13 melanotic lesions had an SBR less than 1 for CLI, despite an SBR greater than 1 among all lesions for beta-RLI. CONCLUSION: beta-RLI can produce functional images of both amelanotic and melanotic tumors in a shorter time frame than CLI. Further engineering developments are needed to realize the full clinical potential of this modality.