Influenza hemagglutinin (HA) and GP64 of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus induce strikingly different initial fusion pores when mediating fusion between host cells that express these fusion proteins and target cells (Plonsky and Zimmerberg, 1996; Spruce et al., 1989, 1991; Zimmerberg et al., 1994). However, in these experiments, variations in host and target membranes confounded the analysis of the role that major components of the fusion reaction play in determining initial pore characteristics. To determine the contribution of the target cell plasma membrane to the fusion pore phenotype, we studied GP64-induced fusion of stably transfected cells (Sf9(Op1D)) to either red blood cells (RBCs) or Sf9 cells. Initial fusion pores in Sf9(Op1D)/RBC and Sf9(Op1D)/Sf9 cell pairs exhibited the same conductance, and pore flickering was not observed in either combination of cells. This indicates that the target cell determines neither the size nor the reversibility of the initial pore. However, the target cell does influence the kinetics of pore formation. The waiting time between triggering and pore appearance was shorter for Sf9(Op1D)/RBC fusion than for Sf9(Op1D)/Sf9 pairs. No correlation between pore waiting time and conductance was found. This argues against a molecular model that assumes aggregation of the pore wall from a nonfixed number of components as the rate-limiting step in GP64 fusion pore formation.