We have investigated the topologies of Ndh (a plastid complex with NADH dehydrogenase activity) and its NDH-F subunit in thylakoids by trypsin and proteinase V8 digestion of both intact and Triton X-100-permeabilized barley thylakoids and identification of the products with antibodies against specific sequences of the NDH-A, NDH-K and NDH-F subunits. Antibody binding and protection against proteinases were also assayed. The analysis of the digestion products of NDH-F by immunodetection and matrix-assisted laser-desorption ionization–time-of-flight allowed us to propose its membrane topology and to compare it with bioinformatic predictions and with that of the homologous subunit (ND5/NuoL/NQO12) of the respiratory complex I. Results indicate that the thylakoid Ndh complex may have an L-shaped structure, similar to that of respiratory complex I, with the hydrophilic arm orientated towards the stroma and the hydrophobic arm inserted into the thylakoid. NDH-A and NDH-K may be located at the bridge between the two arms. Similar to ND5/NuoL/NQO12 of complex I, NDH-F must be distally located in the hydrophobic arm. NDH-F would include up to 15 transmembrane helices and 14 hydrophilic regions. A conserved His-349 in the X transmembrane helix could be involved in H+ pumping. The conserved Thr-181 NDH-F, whose probable phosphorylation increases the activity of the Ndh complex, is located within the hydrophilic region between the V and VI transmembrane helices.