This study aimed to verify the process of lignification in callus of Eucalyptus grandis W. Hill ex Maiden developed amid Murashige and Skoog, 1962 (MS) with different concentrations of growth regulators: 2.4 dichlorophenoxyacetic acid (2,4-D) (4.53, 11.32, 22.65, 45, 30; 90.60 mM) and kinetin (KIN‐6‐furfurilaminopurina) (0, 2.32, 4.65, 11.62, 23.25 mM). The experimental design was completely randomized to five replicates, being each experimental unit consisted of five bottles, containing three segments clonal each one, adding up to 25 treatments. The results were statistically evaluated and indicated that higher concentrations of lignin were associated with a higher concentration of 2.4-D and kinetin on the 2.4-D, when used in the absence of kinetin, at lower concentrations, may stimulate a more intense lignification than when used in the absence of kinetin in higher concentrations. The results indicated also that the calli which are much less oxidized and friable, are related to treatments where the concentration of lignin is of medium to high.