The electronic properties of vemurafenib (VB) provide a rational basis for understanding its strong UVA-induced phototoxicity. Thus, solvation of hydrophobic VB by hydrogen bonding solvents controls its photophysical, photochemical and photosensitizing properties. Addition of phosphate buffered saline (PBS) to methanol (MeOH) induces a bathochromic shift of the VB absorbance spectrum and a fluorescence emission (λmax = 450 nm, quantum yield (Φ) = 0.011). Phosphorescence (λmax = 461 nm) is observed at 77 K in MeOH. 308 nm laser flash spectroscopy demonstrates that the lifetimes (τ) and quantum yields of the VB triplet state ((3)T(*)(1)) in deaerated MeOH (τMeOH = 0.41 μs, λmax ∼ 380 nm), MeOH-PBS and HSA solutions markedly depend on the microenvironment. A long-lived radical (half-life >200 μs) is also formed. The state (3)T(*)(1) is quenched by O2 and electron donors (Cys and 2'-deoxyguanosine) at a rate constant >1 × 10(9) M(-1) s(-1). UVA-irradiation of VB in air-saturated MeOH or MeOH-PBS solutions produces a UVA-absorbing photoproduct (Φ ∼ 5 × 10(-4)). VB photosensitizes Trp destruction by type I (radical formation) and type II (singlet oxygen ((1)O2) formation) photodynamic reactions (Φ = 0.005). Singlet oxygen production is further demonstrated by the VB-photosensitized His oxidation (ΦMeOH = 0.006).