Hydrogen interactions with different microstructural defects were analysed in ultra-low carbon steel, with specific focus on the influence of carbon distribution. For this purpose, the steel was cold rolled and subjected to various annealing treatments, obtaining microstructures ranging from as cold rolled, over recovered up to fully recrystallized. Optical microscopy, transmission electron microscopy and hardness measurements were used to obtain information on the grain boundary structure and dislocation den- sity. Positron annihilation spectroscopy measurements revealed metastable open volume defects related to both dislocations and vacancy clusters. The carbon distribution was characterized by internal friction experiments. Hydrogen interactions were studied by thermal desorption spectroscopy and internal fric- tion measurements of samples electrochemically pre-charged with hydrogen. The most dominant contribution in hydrogen trapping in the cold rolled material is provided by dis- locations. However, their contribution is strongly reduced after annealing at temperatures in the range between 300 K and 600 K due to dissolution of metastable kink-pairs and small carbon-vacancy clusters. Dissolution of such clusters provides fresh supply of carbon to dislocations, which reduces the dislocation trapping capacity for hydrogen due to carbon-hydrogen repulsion. The presence of carbon also reduces the vacancy mobility, allowing clustering and growth during cold rolling resulting in strong hydrogen trapping sites. Binding energies at dislocations were obtained from thermal desorption spectroscopy and internal friction measurements and compared to various models. The small discrepancy in the activa- tion energy is argued to originate from the quantum effect. Hydrogen release from vacancy clusters is determined by the energy required for complete cluster dissolution.