Highly-purified peroxisomes from the yeast Saccharomyces cerevisiae grown on oleic acid were investigated for the presence of channel (pore)-forming proteins in the membrane of these organelles. Solubilized membrane proteins were reconstituted in planar lipid bilayers and their pore-forming activity was studied by means of multiple-channel monitoring or single-channel analysis. Two abundant pore-forming activities were detected with an average conductance of 0.2 and 0.6 nS in 1.0 m KCl, respectively. The high-conductance pore (0.6 nS in 1.0 m KCl) is slightly selective to cations (P(K+)/P(Cl-) approximately 1.3) and showed an unusual flickering at elevated (> +/-40 mV) holding potentials directed upward relative to the open state of the channel. The data obtained for the properties of the low-conductance pore (0.2 nS in 1.0 m KCl) support the notion that the high-conductance channel represents a cluster of two low-conductance pores. The results lead to conclusion that the yeast peroxisomes contain membrane pore-forming proteins that may aid the transfer of small solutes between the peroxisomal lumen and cytoplasm.