We investigated the uptake of imipramine (IMP) in highly purified lysosomes from rat liver and its inhibition by a variety of basic drugs in vitro. The uptake of [(3)H]IMP into lysosomes peaked in less than 20 s, showing little temperature dependency or countertransport phenomena. It was accelerated by increase of extralysosomal pH, stimulated by Mg(2+)-ATP in KCl buffer, and suppressed by acidic ionophores. However, the uptake of [(3)H]IMP in lysosomes was approximately 140-fold higher than the value expected from the pH-partition theory. IMP and other weak lipophilic bases like chlorpromazine and propranolol raised the intralysosomal pH, and their potency was stronger than that of NH(4)Cl, a typical pH-perturbing weak base. A variety of basic drugs inhibited the uptakes of [(3)H]IMP and [(14)C]methylamine into lysosomes, their 50% inhibitory concentrations (IC(50)) being almost the same for [(3)H]IMP and [(14)C]methylamine uptake (r = 0.842). A high correlation (r = 0.946) was observed between the IC(50) values (for the inhibition of [(3)H]IMP uptake) and the lipophilicity (P(oct) values). These results suggest that the accumulation of lipophilic basic drugs is driven primarily by the transmembrane pH difference (pH-partition theory) but with the involvement of some additional mechanism(s) related to drug lipophilicity, possibly binding (partition or adsorption) to lipophilic substance(s) and/or aggregation within lysosomes. Based on this idea, we have established a model that described and successfully simulated the weak base-induced pH increase, the accumulation of a lipophilic weak base (IMP), and the inhibition of accumulation of IMP by lipophilic basic drugs.