To identify the characteristic microstructural length determining the mechanical properties of a quenched and tempered medium-C steel and its dependence on the prior austenite grain size, different tempering treatments have been carried out after a fully martensitic quenching. The resulting microstructures have been analyzed by Orientation Imaging Microscopy (OIM) and two kind of features have been taken into consideration: packets (i.e. domains delimited by high-angle boundaries) and cells (domains bounded by low-angle grain boundaries). The main results can be summarized as follows: 1. A very weak effect of austenite grain size on packet size was found. 2. A finer packet size was measured at mid-thickness with respect to surface after external and internal water quenching process. This phenomenon was attributed to the effect of the strain path on the phase transformation during quenching. 3. The through-thickness microstructural gradient remains substantially unchanged after tempering. 4. Grains with high-angle boundaries do not significantly grow after tempering; on the contrary, low-angle grain boundaries move, fully justifying the hardness evolution with the tempering temperature.