The photophysical properties of seven tetracycline derivatives (tetracycline, oxytetracycline, demeclocycline, chlortetracycline, doxycycline, minocycline and meclocycline) in organic solvents and aqueous solution were studied using steady-state absorption and fluorescence techniques and transient absorption spectroscopies with nanosecond and femtosecond time resolution. The molecular structure, solvent and pH effects on the optical properties of this class of pharmaceutically interesting compounds were investigated in detail. The investigation furnished a complete description of the nature, the spectral and kinetic properties of the excited states formed upon irradiation. All the tetracycline derivatives exhibited a similar behaviour, and the photophysics of these molecules is different in organic solvents and in aqueous medium, where they exhibit a significant pH dependence. In water, compared to organic solvents, these compounds showed a blue-shifted bathochromic absorption band, a red-shifted emission spectrum, an increased Stokes shift and a decreased fluorescence quantum yield. These findings, together with the overall investigated solvent effect, suggested that in aqueous solvent additional fast and non-radiative deactivation processes, responsible for the large Stokes Shift and for the reduced fluorescence efficiency, are present. In fact, in organic media just two transients were observed during the ultrafast time-resolved investigation: the vibrationally hot S1 state which was quickly stabilized by solvent reorganization to the relaxed S1 state. This state showed lifetimes of tens of picoseconds and relaxed by fluorescence and internal conversion. No longer-lived transients were detected. In aqueous solution the excited-state deactivation of tetracyclines was found to be more complicated. Different protonated and tautomeric forms of the S1 state were detected: a component which showed decay times of tens of picoseconds and a component which was longer-lived. A significant pH effect on the nature and number of these components was found. In fact, a remarkable change in the Stokes shift and in the fluorescence efficiency was also observed on going from acidic to basic aqueous solutions. The most important variations in the absorption properties were found in the pH range in which the second acid–base equilibrium takes place. The tetracycline lowest excited triplet state was observed as a ‘rest absorption’ during the femtosecond-resolved measurements in aqueous solution; through the nanosecond-resolved laser flash photolysis study, lower-energy radical species were detected, characterized by lifetimes of tens of microseconds. The formation of these species may be involved in the observed phototoxicity of the tetracycline drugs.