Senescence-induced alterations in photosystem II (PS II) structure and photofunctions were probed in cucumber (Cucumis sativus) cotyledons, using fast O-J-I-P Chlorophyll a (Chl a) fluorescence transients. Analysis of measured and derived parameters of the fast fluorescence O-J-I-P transient revealed senescence-induced alterations in (i), PS II acceptor side electron transfer equilibrium between QA and QB, the primary stable and secondary acceptors of PS II; (ii), intersystem PQ pool size and (iii), affected electron transfer from PS II to PS I. Also, senescence of cotyledons triggered conversion of QA-reducing (fully active) to non- QA-reducing PS II (heat sink) centres. Further, some of the remaining active PS II centres showed a high apparent trapping efficiency due to clustering and energetic connectivity (grouping) between the antennae of active and inactive centers. The overall density of active PS II reaction centers showed a temporal decrease due to the onset of foliar senescence. Thus, the fast Chl a fluorescence transients, with a time resolution of at least 50 mircosec and use of the equations of JIP-test, provide a valuable, non-invasive rapid biophysical probe to study the ageing in plants in terms of detecting photosynthetic activities and the heterogeneity of different types of photosynthetic units. Further, these results were found to be in agreement with the earlier in vitro studies using thylakoids isolated from senescing cotyledons where it was shown that senescence induced heterogeneity in PS II centers affected acceptor side QA<-->QB equilibrium.