The aim of this work was to estimate the changes in contents of different sulfur (S) fractions in soils under lowering inputs of S from emissions together with the influence of organic and mineral fertilizing. Soil samples from long-term field experiments were used for this purpose. The samples were taken from 10 sites from precise long-term field experiments with different soil-climatic conditions in the Czech Republic. The samples were analyzed using the following fractionation: i) water soluble S (H2O extracts), ii) sorbed S (0.032M NaH2PO4 extracts) and iii) S occluded with carbonates (1M HCl extract). Furthermore, the concentration of total S (Stot) and organic S (Sorg) was determined. Soil samples were taken in the years 1981 and 2007. During 26 years a decreases of Stot by about 3-8%, water soluble S by 65-68 % and sorbed S by 39-44 % was observed in the topsoil of the evaluated soils. Furthermore, a low increase in the organic S content (Sorg) was observed. The estimated ratio of Sorg reached 78.7-80.9% from Stot in the year 1981 and 87.7-89.8 % in 2007. Farmyard manure (40 t.ha-1) applied every 4 years did not have a significant influence on S fractions and Stot contents in soils. Intensive S fertilizing increased Stot and mobile S forms contents in the soils.