Abstract We report on a non-perturbative determination of the parameters of the lattice Heavy Quark Effective Theory (HQET) Lagrangian and of the time component of the heavy-light axial-vector current with N f = 2 flavors of massless dynamical quarks. The effective theory is considered at the 1/m h order, and the heavy mass m h covers a range from slightly above the charm to beyond the beauty region. These HQET parameters are needed to compute, for example, the b-quark mass, the heavy-light spectrum and decay constants in the static approximation and to order 1/m h in HQET. The determination of the parameters is done non-perturbatively. The computation reported in this paper uses the plaquette gauge action and two different static actions for the heavy quark described by HQET. For the light-quark action we choose non-perturbatively O(a)-improved Wilson fermions.