In this study, the direct agglutination test (DAT), using 2-mercaptoethanol (2-ME), kaolin or N-acetylcysteine (NAC) as sample diluents, was used to assay 89 samples from visceral leishmaniasis (VL) patients and 130 samples from patients with other diseases and healthy individuals. Maintaining a cut-off of 1:100, the DAT assays with 2-ME, kaolin or NAC presented sensitivities of 94.4%, 95.5% and 100% (P = 0.09) and specificities of 99.2%, 100% and 97.7% (P = 0.17), respectively. Based on these results, we suggest that NAC can be used as a replacement for 2-ME in the DAT, increasing biosafety in the diagnosis of VL.