Summary The potential of glycerol for long-term preservation of the direct agglutination test (DAT) antigen was evaluated at a fluctuating laboratory temperature of 25–37 °C and at constant temperatures of 37 and 45 °C for a period of 222 days. DAT titres recorded for the three antigen aliquots preserved in 50% (v/v) glycerol and stored at 25–37, 37 or 45 °C at 11 time intervals were within the same range of the control antigen kept at 4 °C. Performance of the glycerol-preserved antigen stored at 45 °C was compared with that of a freeze-dried version on 24 visceral leishmaniasis (VL) and 54 non-VL patients. For all non-VL patients, a maximum DAT titre of 1/800 was recorded for either of the two antigens. For all VL patients, in comparison with the freeze-dried, the glycerol-preserved antigen always had equal or higher titre; in 16 of the 24 VL sera tested, the latter antigen scored three- to sixfold higher titres. As this glycerol preservation method is economical and easy to perform, it has better potential for wider-scale application than freeze-drying.