Resumo. Due to the great area occupied with Pinus sp. plantations in southern Brazil, it is considered necessary to evaluate the impact of this kind of crop on soil organic carbon stock, specially related to fractions from humic substances (HS). On this context, this work aims to evaluate the changes on humic substances quantity and the proportion of organic matter on representative soil of the highlands in Santa Catarina state due to replacement of natural grassland by Pinus taeda stands. Four situations of soil use on areas located in Ponte Alta town were evaluated, being one of natural grasslands(CN) and tree of Pinus taeda forests with 5 (P05), 16 (P16) and 21 (P21) years old on second rotation, on soil classified as Humic Dystrupept. Soil samples composed by seven subsamples were collected on seven points inside every area in the 0-5 cm, 5-10 cm, 10-20 cm, 20-30 cm, 30-40 cm layers. The carbon contents of HS in the SOM were determined by chemical fractionating resulted from alkaline or acid extraction. The HS contents and proportions varied according to the soil use and depth analyzed, showing more quantity in the surface layer and its increasing with pinus age. In general, the highest levels of C on fractions and layers evaluated were observed on P21 and the lowest on P05 uses.