Mannheimia haemolytica is one of the most effective bacteria causing pneumonia in animals. Since antibodies against leukotoxin, which is the most important virulence factor of the agent, play an important role in resistance to disease, selection of strain and medium that produce leukotoxin is most important in vaccination studies. For this purpose, 4 different strains were tested in 4 different culture media tested in the study. Based on the viability counts in tested media, RPMI media was found significantly different from the rest of media (P<0.001). On the other hand, BHIB, BHIB+5% serum and BHIB+%1 Yeast extract media were not found statistically different from each other’s (P>0.05) for the viability counts. However, the viability counts measured after 24 hours of culture of tested strains were found to be highest in medium containing BHIB + 5% horse serum. There were no significant differences among strains related to bacterial counts in tested media (P>0.05). RPMI media was significantly different from the rest of media regarding to amount of produced leukotoxin in direct ELISA (P<0.001). All the strains produced more leukotoxins in RPMI media compared to other 3 tested media. According to our results, the strain MH05 produced the highest amount of leukotoxin followed by MH06. There was no significant difference between MH03 and MH04 strains in this regard. It was thought that there could be negative correlation between viability count of the bacteria and the produced leukotoxin amount in tested media. As conclusion, it was assumed that stress factors like starvation or iron restriction might cause Mannheimia haemolytica to produce more leukotoxin and possibly other virulence factors.