To provide a complete molecular description of the fundamental mechanisms necessary for cell life, a series of studies on different organisms are now undergoing to characterize the whole pattern of proteins expression. Different Proteome projects have been activated in the world and their interest grows together with the increased availability of new genetic data. The scientific community realized that, once the entire genome of an organism have been sequenced, to understand the molecular machinery regulating its cellular processes is essential to identify the genes that are really expressed in each cell, tissues and/or biological fluids and the final structure of the functionally active polypeptide chains. No data have been reported before on the pattern of expression of the proteins in bovine tissues and/or biological fluids, although a number of nucleotide sequences (almost 5000) was deposited in databases. Aim of our investigation was to compensate this gap. A series of studies was performed on Holstein Friesian bovines, the most diffused breed in the world. Different tissues and biological fluids have been homogenized and the protein content was extracted according to conventional procedures. Two dimensional electrophoresis (2D-PAGE) experiments allowed the separation of the thousands polypeptide components occurring in each sample. On this basis, high resolution 2D-PAGE reference maps have been defined for liver, kidney, skeletal muscle, plasma and red blood cells on healthy animals in basal state. Each protein component was identified by advanced mass spectrometry procedures. These analysis was performed using matrix assisted laser desorption ionization (MALDI) and/or electrospray ionization (ESI) techniques on protein digests eventually solved by micro-chromatography separation.