Parallel Artificial Membrane Permeability Assay (PAMPA) is a method to screen drug candidates for membrane permeability. The objective was to characterize the transport of a model weak base, metoprolol, across a three lipid-component PAMPA system (denoted A-PAMPA, for anionic-PAMPA) and challenge ion pairing as a mechanism for metoprolol transport. A-PAMPA was designed to mimic the lipid composition of the enterocyte's plasma membrane and included 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (PS18:1) as an anionic lipid-component. Metoprolol flux was measured across A-PAMPA, as well as across three other PAMPA systems. Permeability studies were conducted under various conditions, with varying pH, ionic strength, and presence/absence of competing cations. Permeabilities of mannitol and benzoic acid, as model neutral and anionic solutes, were also measured. PAMPA membrane fluidity was inferred from anisotropy measurements in liposomes. Ion pairing between metoprolol and PS18:1 was assessed via NMR. Metoprolol transport across A-PAMPA was dominated by an ion pair-mediated mechanism (i.e. metoprolol-PS18:1 complex), rather than a membrane fluidity-mediated mechanism. Compared to other PAMPA systems, metoprolol permeability across A-PAMPA and PS18:1 was high. Permeability and anisotropy values suggested PS18:1 selectively facilitated metoprolol transport, while neutral lipid did not. Additional studies supporting ion pairing of metoprolol across A-PAMPA showed that a) metoprolol transport was self-inhibited across A-PAMPA but not across neutral lipid PAMPA; b) competing cations reduced metoprolol permeability across A-PAMPA but not across neutral lipid PAMPA; and c) NMR spectrum of a mixture of metoprolot and PS18:1 showed a broadening of some metoprolol peaks, presumably due to metoprolol interaction with anionic lipid. Metoprolol transport across a three lipid-component PAMPA system that contained anionic lipid was facilitated by apparent ion pairing. (c) 2006 Elsevier B.V. All rights reserved.