Bacterial IgA1 proteases are thought to be important virulence factors in respiratory tract infections. These proteolytic enzymes specifically cleave one of several post-proline peptide bonds within the hinge region of human immunoglobulin A1 (IgA1). Human IgA1 was cleaved with an IgA1 protease purified from an isolate of non-typeable Haemophilus influenzae (NTHI) and cleavage products were N-terminally sequenced. Cleavage of human IgA1 produced two different sized Fc fragments and N terminal sequencing of both these fragments revealed the sequence -Thr-Pro-Ser-Pro-Ser. The IgA1 protease could produce this double cleavage or it is possible that the bacterial strain of the NTHI could be producing two different IgA1 proteases simultaneously. However, PCR with sequence-specific primers amplified only one sequence of the IgA1 protease gene confirming that the NTHI isolate contained one iga gene, which would code for one IgA1 protease. Thus, one isolate of NTHI, which contains one iga gene sequence has been found to produce an IgA1 protease that cleaves two peptide bonds within the human IgA1 hinge region at replicate sequences. The iga gene sequence determines the unique cleavage specificity of the NTHI IgA1 protease and affects the inhibition of the protease. Further work will be required in order to design specific inhibitors to this important class of proteolytic enzymes.