M2, an acid-activated ion channel, is an influenza A virus membrane protein required for efficient nucleocapsid release after viral fusion with the endosomal membrane. Recombinant M2 slows protein traffic through the Golgi complex (Sakaguchi, T., Leser, G. P)., and Lamb, R. A. (1996) J. Cell Biol. 133, 733-47). Despite its critical role in viral infection, little is known about the subcellular distribution of M2 or its fate following delivery to the plasma membrane (PM). We measured the kinetics of M2 transport in HeLa cells, and found that active M2 reached the PM considerably more slowly than inactive M2. In addition, M2 delayed intra-Golgi transport and cell surface delivery of influenza hemagglutinin (HA). We hypothesized that the effects of M2 on transport through non-acidified compartments are due to inefficient retrieval from the trans-Golgi of machinery required for intra-Golgi transport. In support of this, acutely activated M2 had no effect on intra-Golgi transport of HA, but still slowed HA delivery to the PM. Thus, M2 has an indirect effect on early transport steps, but a direct effect on late steps in PM delivery. These findings may help explain the conflicting and unexplained effects on protein traffic observed with other perturbants of intraorganelle pH such as weak bases and inhibitors of V-type ATPases.