Possibilities and limitations of the detection of hydroxyl radicals via the conversion of terephthalate (TPA) into the strongly fluorescent hydroxyterephthalate were investigated in order to adapt this method for chlorophyll-containing samples. Using model chemical sources of various reactive oxygen species, we confirmed that TPA detects hydroxyl radicals very sensitively, but is not reactive to either hydrogen peroxide or superoxide radicals. As a new result, we showed that the conversion of TPA to hydroxyterephthalate cannot be induced by singlet oxygen, which may be produced in photosynthetic systems under stress. Until now, the TPA method has not been used in photosynthesis research, so necessary adaptations to minimise the effects of chlorophyll and buffering sugars on hydroxyl radical detection were also explored and optimal conditions for using the method in thylakoid preparations are suggested. Anticipating further plant physiology applications, usefulness of the TPA method was tested in a wider range of pH than reported earlier. To demonstrate that this simple and highly specific method can be used as an alternative approach for the detection of hydroxyl radicals in plant samples, we measured these radicals in isolated thylakoid membranes exposed to 312 nm ultraviolet radiation.