In the present study the hardness of individual alpha ( )-Ti grains in Ti-6Al-4V was measured by nanoindentation using Berkovich tip indenter. Additionally, alpha-case layer was induced by performing isothermal heat treatment at 700 °C in air for 500 hours. The average hardness of the -Ti grains found in the bulk material and in the alpha-case layer were 6.7 ± 0.7 GPa and 9.4 ± 1.4 GPa, respectively. The high hardness of the -Ti grains in the alpha-case layer is due to solid solution strengthening caused by interstitial oxygen diffusion. The thickness of the developed alpha-case layer was estimated metallographically and compared with that measured from a hardness profile performed along the layer. Moreover, electron back-scattered diffraction was used to determine the local crystallographic orientation, the texture of the alloy microstructure, as well as phase fraction changes, where the nanoindentation measurements were performed.