Bilayers were formed at the tip of microelectrodes from a suspension of proteoliposomes derived from wild-type and porin-deficient mutant yeast mitochondria. In both preparations, identical cationic channels of large conductance were recorded. This result rules out any relationship between this channel and the outer membrane voltage-dependent anion channel, the activity of which is carried by porin. The ionic selectivity and the voltage-dependence of the yeast cationic channel suggest that it is related to that recently described in mammalian mitochondria. This hypothesis is further supported by the fact that both channels are blocked by a mitochondrial addressing peptide.