A sensitive, culture-based, microtitration technique has recently been developed for determining parasite burdens in organs recovered from Balb/c mice infected with Leishmania infantum. In the present study, this technique was used to examine the efficacy of three, first-line, antileishmanial agents in reducing parasite burdens and eradicating parasites from target organs in mice. Treatment with meglumine antimoniate (50 mg SbV/kg.day) significantly reduced the parasite burdens in the livers and lungs (by about 10-fold and > 100-fold, respectively) but not those in the spleens. Although use of a higher dose of meglumine antimoniate (200 mg SbV/kg.day) resulted in an even more dramatic reduction in the parasite burdens in the livers, it had no significant effect on the burdens in the spleens. Treatment with amphotericin B (0.8 mg/kg every other day) resulted in significant reductions in the parasite burdens in the livers, spleens and lungs of infected mice. Although low doses of aminosidine (20 mg/kg.day) had no effect, high doses (200 mg/kg.day) resulted in undetectable parasite burdens in the livers, for at least 100 days post-treatment, and marked reductions in burdens in the spleens. These results are consistent with previous data from studies using animal models of visceral leishmaniasis. Thanks to the sensitivity of the technique, culture microtitration revealed that none of the drug schedules achieved the elimination of all parasites in all target organs. The murine model used mimics some important features of HIV/Leishmania infantum co-infections in humans.