We present an experimental study of charge transport through single oligo(phenylene-ethynylene) (OPE3) molecules and anthracene substituted OPE3 (OPE3-AC) contacted with a mechanically controlled break junction. Both molecules are measured using two different methods. In the first approach, we measure the low-bias conductance, while the junction is repeatedly broken and fused. In the second approach, instead of applying a fixed bias, we record current–voltage characteristics. For both approaches the data is fitted to a model describing transport through a single level, and information about the level alignment and the electronic coupling to the electrodes is extracted. We find that the electronic coupling is similar for both molecules, but that the level alignment differs. The observed trends are confirmed by quantum chemistry calculations.