Aims: Quantification of the effects of pH, temperature and nutrient limitations on the growth and leukotoxin (LKT) production parameters of Mannheimia haemolytica in batch and chemostat culture. Methods and Results: Mannheimia haemolytica strains OVI-1 and PH12296 were grown aerobically in two semi-defined media. In amino acid-limited cultures, the LKT concentration and yield in terms of biomass (Y-LKT/x) were up to eightfold greater than in carbon-limited cultures. Supplementing amino acid-limited chemostat cultures with cysteine, glutamine, ferric iron and manganese further enhanced the Y-LKT/x values up to threefold. Supplementation of an amino acid-limited batch culture of M. haemolytica strain OVI-1 with these nutrients resulted in an LKT concentration of 1.77 g l(-1) that was 45-fold greater than that obtained in RPMI 1640 medium. Aerobiosis enhanced LKT production. High acetic acid concentrations were produced under carbon-sufficient conditions. The highest maximum specific growth rates were recorded in the range of pH 6.8 to 7.8 and 37 to 40 degrees C. Conclusions: An amino acid-limited culture medium greatly improved LKT production in aerobic batch culture, which could be further enhanced by supplementation with cysteine, glutamine, ferric iron and manganese. Significance and Impact of the Study: It was demonstrated that LKT production by M. haemolytica could be dramatically increased through manipulation of the culture medium composition, which could benefit the production of LKT-based vaccines against bovine shipping fever pneumonia.