UV-B, as a component of sunlight, has potentially damaging effects on biomolecules and triggers a photomorphogenic response in plants. UV-B is perceived by UV RESISTANCE LOCUS 8 (UVR8), a β-propeller protein converted from homodimer to monomer upon UV-B absorption. This structural change triggers a signal transduction pathway when the monomer interacts with CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1), leading to transcriptional reprogramming mediated by the transcription factor ELONGATED HYPOCOTYL 5 (HY5). Two of the early induced genes encode REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1 (RUP1) and RUP2, which promote UVR8 reversion from monomer to dimer. SUPPRESSOR OF phyA-101 (SPA) proteins form a complex with COP1 that is stable under UV-B, yet they associate only indirectly, through COP1, with UVR8. In this work, several aspects of UVR8-mediated UV-B signaling were investigated, providing new tools and new insights into the mechanisms underlying the UVR8 signaling pathway, which will be of use for future investigations.