The lysosomotropic amine primaquine has previously been shown to inhibit both 
secretory and recycling processes of cells in culture. We have used a cell-free 
assay that reconstitutes glycoprotein transport through the Golgi apparatus to 
investigate the mechanism of action of primaquine. In this assay, primaquine 
inhibits protein transport at a half-maximal concentration of 50 microM, similar 
to the concentration previously reported to disrupt protein secretion in 
cultured cells. Kinetic analysis of primaquine inhibition indicates that its 
point of action is at an early step in the vesicular transport mechanism. 
Primaquine does not inhibit the fusion of vesicles already attached to their 
target membranes. Primaquine irreversibly inactivates the membranes that form 
transport vesicles (donor), but not the membranes that are the destination of 
those vesicles (acceptor). Morphological data indicate that primaquine inhibits 
the budding of vesicles from the donor membranes. Once formed, the vesicles are 
refractile to primaquine action, and their attachment to and fusion with 
acceptor membranes proceeds unimpeded. In addition to illuminating the mechanism 
of action of primaquine, this study suggests that the selective action of this 
agent will make it a useful tool in the study of the formation of transport 
vesicles.