Resumo. Pinus tecunumanii is one of the most important tropical species of Pinus in Brazil. This work aimed to develop a micropropagation protocol for Pinus tecunumanii from the apical shoots of young plants grown in a greenhouse. To optimise the aseptic treatment protocol, the effect of 0.05 or 0.1% mercuric chloride in combination with 0 or 1 g L-1 fungicide and NaOCl was evaluated. Next, the effect of 2 or 4 µM BA (6-benzyl adenine) on in vitro multiplication was evaluated over three subcultures. Supplementation with 1.5 g L-1 activated charcoal was tested for elongation. For rooting, the effects of WV5 salts (at half strength) and 20 g L-1 sucrose were evaluated on media supplemented with 2.68 µM naphthalene-acetic acid and 0.44 µM BA. Aseptic treatment of explants with 0.05% mercuric chloride and sodium hypochlorite (with or without fungicide) was effective to establish 83.3% of explants in vitro. On media supplemented with 2 or 4 µM BA, shoot multiplication was promoted. After the third subculture on medium supplemented with 2 µM BA, 70.83% of the explants exhibited new shoots with an average of 3.7 new shoots per explant. On medium containing activated charcoal, elongation was observed in 59.76% of the explants, while on medium without activated charcoal only 33.33% elongated. Rooting was observed in 14.8% of explants on medium supplemented with WV5 salts.