Mannose 6-phosphate receptors carry soluble lysosomal enzymes from the trans Golgi network (TGN) to prelysosomes, and then return to the TGN for another round of lysosomal enzyme sorting. We describe here a complementation scheme that detects the vesicular transport of the 300 kd mannose 6-phosphate/IGF-II receptor from prelysosomes to the TGN in cell extracts. In vitro transport displays the same selectivity observed in living cells in that the transferrin receptor traverses to the TGN at a much lower rate than mannose 6-phosphate receptors. Furthermore, recycling of mannose 6-phosphate/IGF-II receptors to the TGN requires GTP hydrolysis and can be distinguished biochemically from the constitutive transport of proteins between Golgi cisternae by its resistance to the weak base, primaquine.