Using in situ high energy X-ray diffraction study of austenite formation in hypoeutectoid steel with three different initial microstructures (ferrite-pearlite, tempered martensite and bainite), the lattice parameters of ferrite, cementite and austenite are examined on heating at 0.25, 10 and 100 °C/s. The lattice parameters of ferrite, cementite and austenite do not vary linearly with the temperature, especially, in the temperature range where the austenitization takes place. For the austenite, it is suggested that the deviation from the linearity is mainly associated to the carbon content variation. Using Dyson and Holmes equation, the carbon content in austenite is evaluated for any moment of the austenite formation for each initial microstructure and all heating rates. For the ferrite-pearlite microstructure heated at 0.25 °C/s, the carbon content in austenite after complete cementite dissolution corresponds to that of pearlite. Moreover, a rapid decrease in carbon content in the austenite is observed during the first stage of the austenitization (simultaneous dissolution of ferrite and cementite) followed by a slow further decrease during the transformation of the remaining ferrite. The obtained results are discussed using thermodynamic calculations.