Ripening of climacteric fruits is a complex physiological phenomenon, which makes berries attractive/palatable and also determines their nutritional characteristics. We report here a comparative proteomic investigation on tomato fruits from a regional and commercial elite ecotype during maturation. Several hundreds of protein components were resolved on 2-DE gels (pH range 4–7) stained with colloidal Coomassie; almost 57% presented overlapping gel coordinates between the two compared varieties. Specific proteins were recognized in each ecotype as differentially expressed during ripening. Constant and variable components were excised, in-gel digested and analysed by MALDI-TOF-MS and µLC-ESI-IT-MS/MS approaches. Peptide MS and MS/MS data were searched against publicly available protein and EST databases, and 83 protein spots were unambiguously identified by MS. Gel matching procedures allowed further identification of proteins between ecotypes. In general, common variably expressed proteins in both ecotypes during maturation were associated to important physiological processes such as redox status control, defence, stress, carbon metabolism, energy production and cellular signalling. Protein components differentially expressed in each variety were also identified. The role of some of the identified proteins in the berries is discussed in relation to tomato fruit ripening. Accordingly, this investigation provides the first characterization of the tomato fruit proteome and description of its variation during maturation.