The retention behavior of an oligolysine mixture, consisting of two to eight residues, was examined at different concentrations of heptafluorobutyric acid (HFBA) in the mobile phase using a C18 column. A single ion record (SIR) mode of the mass spectrometer produced a distinct retention time for each oligomer component. As the concentration of HFBA increased, the retention time of each oligomer increased. Furthermore, the increase in retention time is chain-length dependent such that, the longer the oligomer chain, the more rapid was the rate that retention time increased. A closed pairing model that presumes an equilibrium between the unpaired state and the paired state with a fixed number of HFBA molecules was used to analyze the retention factor as a function of [HFBA]. Curve fitting gave estimates of the ion-pairing equilibrium constant (K(ip,m)), the distribution constant of paired oligolysine (K(D,ip)), and the number of paired HFBA for each oligolysine (n). The plot of the fraction of paired oligolysine in the mobile phase, estimated from K(ip,m) and n as a function of [HFBA], revealed a cooperative effect. In contrast, an open pairing model that assumes independent pairing of HFBA with each residue failed to describe the observed retention behavior.