Summary—The influence of culture duration on some characteristics of hepatocyte subcellular membranes involved in endocytosis was investigated. Activity of enzymes located in plasma membrane, Golgi apparatus and lysosomes increases with time. These modifications are accompanied with several changes in sedimentation properties of these organelles. Endocytosis of [14C]sucrose and [14C]surcose‐LDL is not affected by culture age. On the contrary, [14C]sucrose‐ASF endocytosis strongly decreases in these conditions. These modifications are delayed to some extent by lowering the temperature. Addition to the culture medium of 3‐methyladenine (an inhibitor of autophagy), sodium butyrate, dimethylsulfoxide, phenobarbital or nicotanamide does not prevent the decrease of ASF endocytosis caused by culture duration. These results indicate that one must be cautions when extrapolating to liverin vitro, observations on endocytosis obtained with primary culture of hepa