The role of chloride ions in regulated secretion is well described but remains poorly characterised in the constitutive system. In the liver, newly synthesised proalbumin is transported to the trans Golgi network where it is converted to albumin by a furin protease and then immediately secreted. We used this acid-dependent hydrolysis and the measurement of specific protein secretion rates to examine the H+ and Cl- ion dependence of albumin synthesis and secretion, a major constitutive protein secretory event in all mammals. Using permeabilised primary rat hepatocytes we show that ordinarily chloride ions are essential for the processing of proalbumin to albumin. However Cl- is not required for transport which continues but releases solely proalbumin. Prior treatment of the cells with Tris (used as a membrane-permeable weak base to neutralise Golgi luminal pH) both eliminated the formation of albumin and very greatly reduced secretion. After washing out Tris, both authentic secretion and processing could be restarted if Cl-, ATP, GTP, cAMP, Ca2+ and cytosolic proteins were added. Hence a requirement for chloride in transport, in addition to processing, can be uncovered by first neutralising pH gradients. Furthermore, the chloride channel blocker DIDS (4,4-diisothiocyanostilbene 2,2-disulphonic acid) reversibly inhibited the constitutive secretory pathway. However, the total mass of proalbumin detectable in DIDS-treated cells fell to 36% of control while the fraction processed to albumin remained almost constant. This clearly dissociates a large part of the Cl- requirement of the constitutive protein secretory pathway from the function of known liver Golgi Cl- channels.