The microstructural characteristics of the hot-worked and subsequently tempered tool steel grade X38CrMoV5-1 was studied as a function of the cooling rate using transmission electron microscopy and three-dimensional atom probe. According to the continuous cooling transformation diagram different cooling rates were chosen to adjust a fully martensitic or mixed microstructure consisting of martensite and bainite. The sample with the highest cooling rate exhibited a martensitic structure with nanometre sized secondary hardening carbides of the type M3C, M2C, M7C3, and MC. M3C and M2C were not stable and transformed to M7C3 as the cooling rate decreased. Furthermore, with decreasing cooling rates an increasing number of M7C3 precipitates are particularly present at former austenite grain boundaries as well as martensite and bainite lath boundaries, which strongly affects the mechanical properties.