In concert with irreversible non-equilibrium peptide translation by the ribosome, the nascent polypeptide chain may integrate into the membrane or translocate to the other side of the membrane, facilitated by the conserved protein translocation channel SecYEG in bacteria and Sec61 in eukaryotes. Assuming equilibrium for the decision processes yielded the biological hydrophobicity scale, reflecting free-energy differences ΔG between the pore interior and membrane. Yet kinetic effects and molecular dynamic simulations suggested that a nascent polypeptide could not sample the two separate environments a sufficient number of times for partitioning in equilibrium. Here we tested the hypothesis employing purified and reconstituted SecYEG harboring a stalled ribosome nascent chain (RNC). The SecYEG-RNC complex was open in a de-energized membrane, allowing ion flow. Application of a membrane potential closed the channel if nascent chain hydrophobicity permitted membrane integration. Taking the ratio of steady-state to initial ion conductances as a measure of nascent chain hydrophobicity, we found delta G for KvAP's voltage sensor (4th helix harboring four arginines) and FtsQ's transmembrane helix to be equal to 0.3 and -2.1 kcal/mol, respectively. Thus, delta G observed in our minimalistic system agrees very well with the position-dependent amino acid contribution of the biological hydrophobicity scale. Characteristic sampling times of ~2 s appear sufficient to reach a steady state for a ~20 amino acid-long segment invalidating the hypothesis of insufficient sampling.