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The extended cleavage specificities of two hematopoietic serine proteases originating from the ray-finned fish, the spotted gar (Lepisosteus oculatus), have been characterized using substrate phage display. The preference for particular amino acids at and surrounding the cleavage site was further validated using a panel of recombinant substrates. For one of the enzymes, the gar granzyme G, a strict preference for the aromatic amino acid Tyr was observed at the cleavable P1 position. Using a set of recombinant substrates showed that the gar granzyme G had a high selectivity for Tyr but a lower activity for cleaving after Phe but not after Trp. Instead, the second enzyme, gar DDN1, showed a high preference for Leu in the P1 position of substrates. This latter enzyme also showed a high preference for Pro in the P2 position and Arg in both P4 and P5 positions. The selectivity for the two Arg residues in positions P4 and P5 suggests a highly specific substrate selectivity of this enzyme. The screening of the gar proteome with the consensus sequences obtained by substrate phage display for these two proteases resulted in a very diverse set of potential targets. Due to this diversity, a clear candidate for a specific immune function of these two enzymes cannot yet be identified. Antisera developed against the recombinant gar enzymes were used to study their tissue distribution. Tissue sections from juvenile fish showed the expression of both proteases in cells in Peyer’s patch-like structures in the intestinal region, indicating they may be expressed in T or NK cells. However, due to the lack of antibodies to specific surface markers in the gar, it has not been possible to specify the exact cellular origin. A marked difference in abundance was observed for the two proteases where gar DDN1 was expressed at higher levels than gar granzyme G. However, both appear to be expressed in the same or similar cells, having a lymphocyte-like appearance.