The evolution of intrinsic stress in multilayers and its behavior with growing number of periods and/or during radiation exposure, i.e. thermal treatment, becomes important in many X-ray optical applications. In Ni/C X-ray optical multilayers fabricated by pulsed laser deposition (PLD) at room temperature with typical period thickness d >> 4.0 nm microstructure and intrinsic stress are analyzed with growing period number up to the layer stack delamination. Microstructure of single layers and interfaces and morphology of the total layer stack were investigated by means of transmission electron microscopy (TEM), X-ray reflectometry and diffraction. A clear indication of nano-crystallites was found for the Ni-layers, whereas the carbon layers were always amorphous. The diffraction pattern of a 300 period Ni/C layer stack hint at a hexagonal structure of the Ni crystallites with the (011)-lattice plane parallel to the Si substrate surface. Thus the elastic misfit between the Ni- and C-layers seems to be minimized. To investigate the evolution of stresses in the growing up Ni/C multilayers depth dependent stresses in the Si (100) substrate were analyzed using X-ray results of the stress tensor for Si-crystal of the substrate averaged over different penetration depths (0.6 and 3.6 μm). The found dependence of the substrate stresses on the total layer thickness point at low compressive stresses in the PLD-fabricated Ni/C X-ray optics in the range below -100 MPa at least up to a total layer thickness of 1200 nm (300 periods). An increase of the compressive stresses was obtained only for stacks of more than 300 periods. A delamination of the layer stack in part was observed at 900 periods.