Leishmaniasis is transmitted between mammalian hosts by the bites of bloodsucking vector sand flies. The dose of parasites transmitted to the mammalian host has never been directly determined. We developed a real-time PCR-based method to determine the number of Leishmania major parasites inoculated into the ears of living mice during feeding by individual infected flies ( Phlebotomus duboscqi ). The number of parasites transmitted varied over a wide range in the 58 ears in which Leishmania were detected and demonstrated a clear bimodal distribution. Most of the infected mice were inoculated with a low dose of <600 parasites. One in four received a higher dose of >1,000 and up to 100,000 cells. High-dose transmission was associated with a heavy midgut infection of >30,000 parasites, incomplete blood feeding, and transmission of a high percentage of the parasite load in the fly. To test the impact of inoculum size on infection outcome, we compared representative high- (5,000) and low- (100) dose intradermal needle infections in the ears of C57BL/6 mice. To mimic natural transmission, we used sand fly-derived metacyclic forms of L. major and preexposed the injection site to the bites of uninfected flies. Large lesions developed rapidly in the ears of mice receiving the high-dose inoculum. The low dose resulted in only minor pathology but a higher parasite titer in the chronic phase, and it established the host as an efficient long-term reservoir of infection back to vector sand flies.