Quantifying the passage of the large peptide protamine (Ptm) across CymA, apassive channel for cyclodextrin uptake,isinthe focus of this study.Using areporter-pair-based fluorescence membrane assay we detected the entry of Ptm into liposomes containing CymA. The kinetics of the Ptm entry was independent of its concentration suggesting that the permeation through CymA is the rate-limiting factor.F urthermore,w e reconstituted single CymA channelsi nto planar lipid bilayers and recorded the ion current fluctuations in the presence of Ptm. To this end, we were able to resolve the voltage-dependent entry of single Ptm peptide molecules into the channel. Extrapolation to zero voltage revealed about 1-2 events per second and long dwell times,i na greement with the liposome study.A pplied-field and steered molecular dynamics simulations added an atomistic view of the permeation events.Itcan be concluded that ac oncentration gradient of 1 mm Ptm leads to at ranslocation rate of about one molecule per second and per channel.