Expressed Sequence Tags (ESTs) and cDNA-AFLP analysis have been used to study gene expression during peach fruit ripening. An EST repertoire (1007 sequences) has been constructed using transcripts in nectarine fruit cv. 'Fantasia' at climacteric stage. The EST repertoire was reduced to 696 unigenes: in these 579 (83%) were singletons. EST analysis confirmed that in peach ripe fruit, genes involved in ethylene biosynthesis and cell wall metabolism are highly represented. Three transcription factors (TFs) homologous to SEPALLATA3, atb2, bHLH61 belonging to MADS, bZIP and bHLH families, respectively, have been identified. The abundance of the related transcripts suggests that these TFs play a regulatory role of ripening, as reporter in other fruits species. The EST repertoire has been included in a sequence database assembled by the Italian Consortium for Prunus spp genomic study to generate the first peach fruit microarray (μPEACH 1.0). EST analysis has been integrated with cDNA-AFLP to study a selection of the nectarine cv. 'Fantasia', named sir, characterized by a block of ripening. Data pointed out that the sir fruit phenotype might be due to disturbance in the onset of senescence.