The formation of the fusion pore is the first detectable event in membrane fusion (Zimmerberg, J., R. Blumenthal, D.P. Sarkar, M. Curran, and S.J. Morris. 1994. J. Cell Biol. 127:1885-1894). To date, fusion pores measured in exocytosis and viral fusion have shared features that include reversible closure (flickering), highly fluctuating semistable stages, and a lag time of at least several seconds between the triggering and the pore opening. We investigated baculovirus GP64-induced Sf9 cell-cell fusion, triggered by external acid solution, using two different electrophysiological techniques: double whole-cell recording (for high time resolution, model-independent measurements), and the more conventional time-resolved admittance recordings. Both methods gave essentially the same results, thus validating the use of the admittance measurements for fusion pore conductance calculations. Fusion was first detected by abrupt pore formation with a wide distribution of initial conductance, centered around 1 nS. Often the initial fusion pore conductance was stable for many seconds. Fluctuations in semistable conductances were much less than those of other fusion pores. The waiting time distribution, measured between pH onset and initial pore appearance, fits best to a model with many (approximately 19) independent elements. Thus, unlike previously measured fusion pores, GP64-mediated pores do not flicker, can have large, stable initial pore conductances lasting up to a minute, and have typical lag times of < 1 s. These findings are consistent with a barrel-shaped model of an initial fusion pore consisting of five to eight GP64 trimers that is lined with lipid.