The intracellular translocation and processing of epidermal growth factor (EGF) in 3T3 cells has been studied utilizing Percoll density gradients. EGF is internalized and rapidly becomes associated with two types of intracellular compartments. The extent to which EGF is delivered to these two compartments is apparently regulated depending upon the cell's physiological condition. In growth medium, an increased proportion of EGF is taken up into a Golgi-like element. Uptake through this pathway correlates with a decrease in degradation of the ligand. In the absence of serum and amino acids, an increased proportion of EGF is taken up into a component which has a density of 1.05. Uptake through this pathway correlates with increased degradation of the ligand. The ligand taken up through both pathways is transferred to dense vesicles which comigrate with lysosomes. In the presence of growth medium, however, dense vesicles containing EGF can be shown to be lysosomal enzyme-deficient upon further fractionation. In addition, in the presence of serum, a portion of the internalized EGF is apparently released from the cells, intact, and then re-bound. The processes described may be important in the production of a mitogenic response and the ability of cells to self-regulate their responsiveness to the growth factor.