We report on experimental high-resolution spectroscopic studies in combination with advanced theoretical calculations that focus on the excited-state dynamics of various forms of the chromophore of the Photoactive Yellow Protein (PYP), and the dependence of these dynamics on conformational and isosteric structure, as well as the biological environment. Three-colour nanosecond multiphoton ionization pump–probe studies confirm and extend previous conclusions that the dominant decay channel of the lowest excited ππ* state (the so-called V′ state) of methyl-4-hydroxycinnamate is picosecond internal conversion to the adiabatically lower nπ* state, and enable us to resolve apparent contradictions with picosecond pump–probe studies. Comparison of multiphoton ionization and laser induced fluorescence excitation spectra leads to the assignment of the hitherto elusive excitation spectrum of the V(ππ*) state. Complexation of methyl-4-hydroxycinnamate with water radically changes the excited-state dynamics; internal conversion to the nπ* state is absent, and bond isomerization channels instead play a prominent role. Excited states of the thio-ester compound, the form in which the chromophore is present in PYP, have till the present study remained out of reach of gas-phase studies. The excitation spectra obtained here show a broad, almost structureless band system, giving evidence for enhanced nonradiative decay channels. The gas-phase results will be discussed in the context of results from ultrafast studies on these two chromophores in solution.