The infectivity of influenza viruses to host cells depends on the activation of the viral glycoprotein hemagglutinin (HA) by proteases. Starting from the observation that influenza virus replication in MDCK (Madin Darby canine kidney) cells was impaired by inactivation of trypsin in the culture fluids, we demonstrated that the inhibitory activity was resolved into two Trypsin-inactivating factors (TF), TF A (15 kDa) and TF B (11 kDa). N-terminal protein sequences of the factors revealed that TF A was a known Submandibular Protease Inhibitor (SPI) secreted in dog saliva, while TF B was a novel protein (renamed CKPI; canine kidney protease inhibitor). Following peptide mapping and protein sequencing of CKPI we obtained a 390 bp cDNA encoding a 130-amino-acid protein from MDCK cell total RNA. Protein sequence comparison showed a 63.8% identity with human secretory leukocyte protease inhibitor (SLPI), the molecule containing two conserved whey acidic protein (WAP) motifs, and we suggest that CKPI is thought to be the canine analogue of human SLPI. These results suggest that the inhibitory factors are secreted from MDCK cells, which are involved in prevention of virus replication, and applicable to the protection of host cells from virus infection.