The Penning ionization (PeI) source uses atoms of rare gases or molecules (N2) excited to give a flux of metastable atoms or molecules (A*) able by collision to ionize a target molecule (M) on the condition that the process is exothermic (i.e., IE(M) < EE(A)). As electron ionization, PeI allows the ionization of apolar species such as saturated hydrocarbons yielding molecular ions. In this work we present the application of vacuum PeI source coupled with a FT-ICR instrument for the characterization of a diesel fuel. Argon and krypton, as metastable gas, allow reducing significantly the fragmentation extent compared to electron ionization. Unlike with an atmospheric pressure source, the use of a vacuum source allows a good control of the ionization conditions with the absence of oxygen or other reactant such as water.