In the endocytic compartment, an acidic pH plays a key role in receptor and ligand sorting, vesicular transport, and protein degradation. In the secretory compartment, indirect estimates of trans-Golgi pH based on partitioning of weak bases and following viral infection suggest a mildly acidic pH of > 6.0. We developed a liposome microinjection method to introduce fluorescent indicators into the aqueous compartment of trans-Golgi in living cells. In the presence of ATP and at 37 degrees C, 70-nm diameter liposomes delivered their fluid-phase contents selectively into the trans-Golgi compartment as assessed by colocalization with the trans-Golgi stain N-[6-[(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino]caproyl]-sphingosine (C6-NBD-ceramide). Liposome fusion was ATP- and temperature-dependent and blocked by N-ethylmaleimide but not by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). trans-Golgi pH in skin fibroblasts was 6.17 +/- 0.02 (S.E., n = 174) as measured by ratio imaging confocal microscopy using fluorescein and rhodamine-based indicators and an in vivo calibration procedure. trans-Golgi pH increased to 6.8 +/- 0.1 by cAMP agonists and to 6.5 +/- 0.1 by protein kinase C activation. These results provide the first direct measurement of trans-Golgi pH in living cells and demonstrate pH regulation by second messengers.