Canine distemper virus (CDV) causes serious and often fatal disease in dogs. Currently, various cells or cell lines have been used to detect or produce CDV. In order to set up the conditions, we separated two different cell lines from Madin-Darby canine kidney (MDCK) and named as MDCK-F (fibroblast-like) and MDCK-E (epithelial-like) by Na2EDDA treatment. CDV seed virus was prepared using MDCK cells and inoculated into MDCK-F and MDCK-E including MDCK with various ranges of multiplicity of infection (MOI) to confirm the optimal amount of virus inoculation. The virus titer of TCID50/ml was calculated by inoculation of serially diluted virus into 96-well plate of MDCK cells. The titer and cytopathic effect (CPE) in MDCK-E were compared to those in MDCK-F. The titer of seed CDV was 1.24×106 TCID50/ml. Optimal MOI was about 0.1 for both MDCK-F and MDCK-E to obtain highest titers of 108 TCID50/ml and 5 × 108 TCID50/ml respectively. CPE in MDCK-E was shown 4 days after inoculation whether in MNCK-F 5 6 days after inoculation. We can obtain highest titer of 5 × 108 TCID50/ml with 0.1 MOI using MDCK-E. MDCK-E was more susceptible for CDV production than MDCK-F.