The principal mechanism of cellular uptake of benzo(a)pyrene and other polycyclic aromatic hydrocarbons (PAH) from lipoproteins into cells is spontaneous transfer through the aqueous phase (Plant, A. L., Benson, D.M., and Smith, L.C. (1985) J. Cell Biol. 100, 1295-1308). Cellular uptake of benzo(a)pyrene from low density lipoproteins followed first-order kinetics with a rate constant that was independent of the relative lipoprotein concentrations or cell number but which was 2 orders of magnitude smaller than the rate constant for benzo(a)pyrene desorption from low density lipoproteins. Moreover, identical rate constants for cellular uptake of benzo(a)pyrene were observed when the donor vehicle was high density lipoproteins, very low density lipoproteins, or single bilayer phosphatidylcholine vesicles, even though rate constants for benzo(a)pyrene transfer from these donor vehicles differed by 10-fold. When phosphatidylcholine vesicles containing benzo(a)pyrene and a nontransferable fluorescence quencher were mixed with cells in a stopped-flow system, two kinetic components were distinguished: a fast component with a rate constant corresponding to that measured for transfer of benzo(a)pyrene out of vesicles, followed by a much slower component, with a time course approximating that measured for cellular accumulation of benzo(a)pyrene by other techniques. Rate constants for desorption of a series of PAH which contained different number of aromatic rings from phosphatidylcholine vesicles differed over a 70-fold range. First-order rate constants for cell uptake of benzo(a)pyrene and five other PAH of different molecular sizes had the same 70-fold range of values, but were 2 orders of magnitude smaller than their respective rate constants for desorption from single bilayer vesicles. In addition, activation energies for cell uptake were essentially identical to the respective activation energies for desorption of PAH from phosphatidylcholine vesicles, confirming the mechanistic similarity of the two processes.