Background: Culture media are often essential for diagnosis of metabolism and antigenic properties of leishmania promastigotes and laboratory studies. Fetal calf serum (FCS) has long been used as a supplement in leishmania culture media. There are many technical problems in FCS processing such as sterilization (especially viral contamination removal) and high cost. In this study growth stimulating effects of brain heart infusion broth (BHIB) on L.major promastigotes culture was assessed. The possibility of FCS replacement with BHIB as an appropriate supplement in single phase media and also as an enhancer for mass culture of L.major promastigotes in biphasic media was also evaluated.Methods: n this study, "RPMI 1640" and "agar and blood agar" were used as single-phase and biphasic medium, respectively. They were supplemented with different concentrations of BHIB for leishmania promastigotes culture. RPMI 1640 containing 10% FCS was used as control medium. Biphasic medium containing normal saline was used to culture parasite promastigotes. The numbers of proliferated promastigotes were determined at definite time intervals and the average numbers of promastigotes in each media were compared with the control medium.Findings: The average number of L.major promastigotes in presence of BHIB 10% in RPMI 1640 was 22.7 × 106 /ml which was significantly higher compared to the controls (P = 0.012). The average numbers of L.major promastigotes in the presence of BHIB 4% in agar and blood agar media were 275 × 106 /ml and 367 × 106 /ml, respectively. These numbers were also significantly higher compared to the control medium (P = 0.025). Conclusion: These results indicated that different concentrations of BHIB have a promoting effect on the proliferation of L.major promastigotes. Therefore, BHIB may be an appropriate substitute for FCS in single-phase media and biphasic media for mass culture.