A potential vaccine candidate for visceral leishmaniasis should favour the development of CD4+ Th1 type of immune response which is further dependent on the dose of antigen and the route of inoculation. The present study was carried out to check the effective dose (low, medium and high) and route (subcutaneous, intradermal, intraperitoneal and intracardiac) of inoculation for the development of a CD4+ Th1 type of immune response in BALB/c mice. The parasite load was found to be the lowest in mice inoculated with low dose of promastigotes through the subcutaneous route, followed by intradermal, intraperitoneal and intracardiac routes. A reduced parasite load in mice inoculated through subcutaneous route was found to be associated with heightened DTH responses. The IgG2a levels were found to be the maximum in case of mice inoculated with the low dose of promastigotes through subcutaneous route followed by intradermal and intraperitoneal routes. In contrast, mice inoculated with high dose of promastigotes through the intracardiac route showed increased levels of IgG1. Low-dose inoculation with subcutaneous route elicited maximum IFN-gamma levels, which points towards the generation of Th1 response. Maximum IL-4 and IL-10 levels were detected in high-dose inoculation through intracardiac route suggesting the development of Th2 response. In conclusion, inoculation through subcutaneous route with low dose of live whole parasite antigen evokes a strong Th1 response in BALB/c mice.