Some polypeptide substrates for spleen fibrinolytic proteinase (SFP) were synthesized and specificities for the enzyme were compared with those for granulocyte elastase (GE). The results obtained were as follows. 1) The synthetic substrate specific for SFP and GE were dominantly characterized by C-terminal Val. 2) Among 8 kinds of stereoisomers of Suc-Tyr-Leu-Val-pNA, L-L-L type and D-L-L type were catalyzed easily by SFP, other types, however, showed inhibitory effect for GE as well as SFP. The lowest Ki value was found in L-D-D type (0.12mM). 3) In the studies on the chain length, the best kinetic constants were found in the substrate having 5 amino acids, i. e. Suc-Ala-Ala-Tyr-Leu-Val-pNA. However, 4 amino acids peptide substrate, Suc-Ala-Tyr-Leu-Val-pNA, which had lower Km than <Glu-Pro-Val-pNA (S-2484, KABI), was concluded to be the most appropriate substrate for SFP and GE in high solubility and specificity so far examined. 4) Substrate specificity of GE was similar to that of SFP in all synthetic peptide substrates used.