The aim of this study is to investigate physicochemical characteristics and in vitro cell interactions of comb-type poly(ethylene glycol) (Mn= 10 700 and Mn= 20 200 g/mol, p(PEG-A) 10K and p(PEG-A) 20K, respectively) in comparison with linear poly(ethylene glycol) (Mn= 10 000 and Mn= 20 000 g/mol, PEG 10K and PEG 20K, respectively) of equivalent molecular weight. In vitro cytotoxicity, cell uptake and intracellular distribution profile of comb-type and linear polymers were investigated using human lung adenocarcinoma epithelial cells (A549). The dynamic light scattering (DLS) analysis showed that the comb-type polymers had smaller hydrodynamic diameters (Dh) (4.5±0.3 nm - 5.9±0.3 nm) than linear PEGs (5.6±0.5 nm - 7.8 ±0.2 nm) in water and phosphate buffer solution at pH 7.4. While the Dh of p(PEG-A) 10K and 20K in RPMI 1640 and RPMI 1640 containing 10% FBS ranged between 44.4±1.8 nm and 58±5.3 nm, the Dh of PEG 10K and 20K in the same media were between 54.5±4.7 nm and 63.5±2 nm. According to the AFM analysis, PEG 10K forms supramolecular linear structures whereas p(PEG-A) 10K forms spherical structures. None of the polymers caused significant cytotoxic effect on A549 cells under the conditions tested via a cell viability assay. Cell uptake studies via flow cytometry showed that the uptake of p(PEG-A) 10K by A549 cells was significantly higher than the other polymers tested. All polymers were internalized by A549 cells via an active transport mechanism. The uptake increased with increasing polymer concentrations. None of the polymers affected the cell cycle of A549 cells under the conditions tested. Both the comb-type and linear PEGs were found to localize in the lysosomes of A549 cells.