2-(3H)-benzoxazolinone (BOA) is a secondary plant metabolite previously found to inhibit plant growth and development. The phytotoxic activity of BOA has been extensively demonstrated over the last years. However, the relation of BOA phytotoxicity with BOA accumulation in plant leaves has not been thoroughly investigated. In this work, BOA phytotoxicity on photosynthesis (PhiPSII and Pn) of lettuce (Lactuca sativa L. cv. Great Lakes) was studied, and these results were correlated with BOA quantities in the leaves. BOA-treated plants showed reduced photosynthesis rate 6 h after the beginning of the treatment, and the efficiency of photosystem II started to be affected 10 h after treatment. These results were correlated with an increasing concentration of BOA in leaves that starts 6 h after treatment and shows a maximum at 96 h.