Abstract: A callogenesis protocol for Melaleuca alternifolia was developed in this study. The effects of different types of auxins, activated charcoal and combination of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin at various concentrations on the callus induction from leaf explants of M. alternifolia were investigated in order to determine the optimum callus induction and maintenance medium. The callus growth curve under the best callus maintenance medium was also studied. Calli were induced from leaf explants cultured on Murashige and Skoog (MS) medium incorporated with 1, 3 and 5 mg L-1 of 2,4-D, Indole-3-butyric Acid (IBA), Naphthaleneacetic acid (NAA) or picloram as well as MS medium with the combination of 3 mg L-1 2,4-D and 1, 2 or 3 mg L-1 kinetin, respectively. The calli developed from different types of plant growth regulators were varied in their morphological appearances. The maximum callogenesis response was obtained using the MS medium supplemented with 3 mg L-1 2,4-D. The control medium and the medium incorporated with NAA did not produce any callus. Addition of activated charcoal at 0.05 and 0.1% did not assist in the callogenic response, though it helped in removing the pigments and phenolic compound accumulations in the culture medium. The calli initiated from the best induction medium could not be maintained in the fresh new medium containing the same auxin composition. However, calli were successfully maintained in MS medium containing the combination of 3 mg L-1 2,4-D and 2 mg L-1 kinetin. The callus maintained in this treatment showed a sigmoid growth pattern and reached maximum the growth rate after three to five weeks upon cultivation.