Voltage clamp conditions were used to study the membrane permeability properties of rough microsomes (RM) derived from the rough endoplasmic reticulum of canine pancreas and inverted vesicles (InV) derived from the plasma membrane of Escherichia coli. Membrane vesicles of RM or InV were fused to a planar lipid bilayer that was formed in a hole of a partition separating two chambers. Fusion of a single RM vesicle yielded a single-step conductance increase. Some preparations yielded unitary conductances of 20, 55, 80, and 115 pS in 45 mM potassium glutamate. These channels were largely open at negative membrane potential on the cytoplasmic side of the RM membrane, mostly closed at positive voltages, permeable to amino acids, and slightly more selective for anions than cations. There was a dramatic increase in the number of open channels when 100 microM GTP was added to the cytoplasmic side of the fused RM, whereas 100 microM guanosine 5'-[gamma-thio]triphosphate caused closing of channels. ATP had no effect. A large channel of 115 pS at 45 mM potassium glutamate was also detected after the fusion of InV. As both RM and InV share the ability to translocate secretory proteins, it is possible that the 115-pS channel in both membranes represents a protein-conducting channel.