Efficient transferrin receptor recycling is reconstituted when donor cytosol and ATP are added to the streptolysin O permeabilized cells. The rate of reconstituted recycling is dependent on the concentration of donor cytosol. The cytosol provides a factor(s) required for the transport of transferrin from the pericentriolar recycling compartment to the plasma membrane. N-Ethylmaleimide treatment of permeabilized cells inhibits both the fusion of recycling vesicles with the plasma membrane as well as the formation of functional recycling vesicles from the pericentriolar recycling compartment. Guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) does not affect reconstituted recycling in the presence of an optimal cytosol concentration. Therefore, the rate-limiting step in recycling is not regulated by GTP-hydrolyzing proteins, and hydrolysis of GTP is not required for endocytic recycling. GTP gamma S stimulates recycling when suboptimal concentrations of cytosol are used. This stimulatory effect is not mediated by a brefeldin A-sensitive ADP-ribosylation factor protein. Addition of wild-type donor cytosol to permeabilized END2 Chinese hamster ovary cells, which recycle transferrin at half the rate of wild-type cells, reconstitutes recycling to the reduced rate of intact END2 cells but not to the wild-type recycling rate. These results indicate that the defect responsible for the slowed transferrin recycling in END2 mutants is membrane associated or that the defective protein is too large to diffuse out of the cells through the streptolysin O pores.