Serine proteases are major granule constituents of cells from several mammalian hematopoietic cell lineages. Despite the relatively extensive knowledge about these mammalian proteases, very little is known about their bird, reptile and amphibian homologs. In order to close this gap in our understanding of the evolution of these proteases, we have characterized the extended cleavage specificity and hematopoietic expression pattern of the chicken serine protease cathepsin G-like. This protease, which clusters in a separate subfamily of serine proteases among the vertebrate hematopoietic serine proteases, has been characterized using substrate phage display and further validated by using a panel of recombinant substrates. A preference for a lysine in the P1 position of a substrate, arginines in positions P2 and P3, and the aromatic amino acid tryptophane in the P4 position was observed. Based on the sequence alignment we could identify a consensus sequence for this protease as being PGGWRRK↓ALSV. Mass spectrometry analysis of a peptide with the consensus sequence obtained by phage display showed that cleavage of this peptide occurred after the conserved Lys (K) residue. A screening of potential in vivo substrates based on the derived P5-P3' consensus sequence resulted in a relatively limited number of potential substrates, due to the high selectivity of this enzyme. The most interesting of these were PDGF-A, coagulation factor V and low-density lipoprotein receptor like-8. Immunohistochemical analysis of chicken white blood cells with antisera produced against chicken cathepsin G-like and chicken egg lysozyme, as a reference protein known to be expressed by hematopoietic cells, showed presence of chicken cathepsin G-like almost exclusively in thrombocytes whereas lysozyme was found at very high amounts in heterophils, and lower amounts in monocytes and thrombocytes.