Changes in dislocation substructure were studied in normalized and annealed S235JR steel in relation to the number of symmetrical reversed stress cycles with the amplitude σa = 242 MPa and corresponding mean life Nf = 17,950 cycles. The microstructure of the steel consisted of ferrite with a small amount of pearlite. The substructure was observed in as received condition and after application of various relative numbers of cycles n/Nf, these being 0.25, 0.50 and 0.75. An irregular dislocation net occurred in the virgin specimen, however, during cyclic loading the dislocations started to accumulate gradually in slip bands and to form a cell substructure in grains of favourable crystallographic orientations. Total dislocation density, dislocation density in slip bands and inter-band distance were measured using transmission electron microscopy (TEM). The total dislocation density was found to slightly decrease and the density in slip bands to increase with increasing number of cycles. The results concerning the dislocation density were compared with changes in the microplastic limit (MPL) which were determined by the measurement of the inductance of the “specimen - coil” system. They consisted in a rapid initial decrease at the first stage of the fatigue process and in a gradual increase during the major part of the life. These changes can be interpreted on the basis of changes in dislocation density as was verified independently by X-ray diffraction and nanoindentation tests. On the basis of the measurement of dislocation density by TEM it appears that changes in MPL can be connected with the dislocation density in slip bands rather than with the total dislocation density.