The induction of channels across planar lipid bilayers by purified, recombinant pneumolysin (a hemolytic protein from Streptococcus pneumoniae) has been studied by measuring increases in electrical conductivity. Pneumolysin-induced channels exhibit a wide range of single channel conductances (less than 50 pS to greater than 1 nS at 0.1 M KCl). Channels can be categorized on the basis of their K+:Cl- selectivity: the smallest channels are strongly cation selective, with t+ (the cation transference number) approaching 1.0; the largest channels are unselective (t+ approximately 0.5). Channels tend to remain open at all voltages (-150 to 150 mV); only the smallest channels exhibit any rectification. In the presence of divalent cations (1-5 mM Zn2+; 10-20 mM Ca2+), small (less than 50 pS) and medium-sized (50 pS to 1 nS) channels are closed in a voltage-dependent manner (more closure at higher voltages); at 0 voltage channels reopen. Overall selectivity is reduced by divalent cations, compatible with small, selective channels being closed preferentially to large, nonselective ones. It is concluded that a single molecular species (pneumolysin) induces multiple-sized channels that can be categorized by cation:anion selectivity and by their sensitivity to closure by divalent cations.