A Nicotiana tabacum Affymetrix-based custom expression array has been developed using EST sequence data generated by Advanced Technologies (Cambridge) (ATC) and Altadis, together with publicly-available data. 46,546 sequences were generated from a collaborative sequencing project carried out by ATC and Institut du Tabac de Bergerac (Altadis), (for further information on this project please see http://www.estobacco.info/index.html). A further 13,555 sequences were produced by ATC. cDNA libraries were synthesised for sequencing from several tissue types at different developmental stages and 4 varieties of tobacco (K326, Burley 21, TN86 and Samsun). A unigene set was compiled (F. Dorhlac de Borne, Altadis) containing 40, 642 sequences (contigs, singletons and published ESTs) and submitted to Affymetrix for probe design. A 64-format, 11 µm feature size expression array was created comprising 43,723 probe sets.The arrays have been used to investigate changes in gene expression in senescing tobacco leaves. Senescence is a dynamic, tightly-regulated event representing the final stage of leaf development and significant metabolic change. It also signifies the onset of 'ripening' in tobacco when leaves are harvested for curing. In this study, darkness was used to uniformly induce leaf senescence. Nicotiana tabacum (var. K326) plants were grown from seed in a greenhouse (22°C and 16 hours of light). At approximately 6-weeks old, 40 plants were transferred to 2 identical growth cabinets set at 22°C; 20 plants were kept in constant darkness and 20 were maintained under normal light conditions. At day 0, 1, 10 and 22, a single leaf (leaf 6 from the base of the plant) was taken from 5 plants in the light and 5 in the dark. These plants were then disregarded from the rest of the study. Leaves were flash frozen, ground in liquid nitrogen and RNA extracted. cRNA was synthesised and labelled using GeneChip^→one-cycle target labelling and control reagent kit and hybridised to the Nicotiana microarray chip according to the Affymetrix Expression Analysis Technical Manual. Chips were scanned and analysed using the GeneChip^→Scanner 3000 with initial QC checks carried out in GCOS. The quality of the data was further assessed using the Expression ConsoleTM software prior to detailed analysis of gene expression in Genespring GX 7.3.1. Statistically significant differences in gene expression between the plants grown in darkness and the control plants were found using a Welch t-test with p-value cut off of 0.05 and Benferroni multiple testing correction. Data was subsequently filtered to reveal genes that showed at least 2-fold up-regulation during dark treatment. Venn diagrams were created to show the commonality of these gene lists between the day 1, 10 and 22 time points. This is, to our knowledge, the first description of an Affymetrix designed expression array for Nicotiana tabacum and will enable further studies of large-scale molecular events in tobacco, an important model organism and commercial crop.