Conus venoms are estimated to comprise over 100,000 distinct pharmacologically active peptides, the majority probably targeting ion channels. Through the characterization of a cytolytic peptide from the venom of Conus mustelinus, conolysin-Mt, we expand the known conopeptide mechanisms to include association with and destruction of cellular membranes. A new 23AA conopeptide, conolysin-Mt has potent hemolytic activity when tested on human erythrocytes. At a concentration of 0.25 microM, the peptide permeabilized both negatively charged prokaryotic (PE:PG) and zwitterionic eukaryotic (PC:cholesterol) model membranes. The affinity constants (KA) of conolysin-Mt for PE:PG and PC:cholesterol model membranes were 0.9 +/- 0.3 x 10(7) and 3 +/- 1 x 10(7) M-1, respectively. In contrast, conolysin-Mt exhibited low antimicrobial activity (MIC > 50 microM) against two Escherichia coli strains, with an MIC for the Gram-positive S. aureus of 25-50 microM. The specificity of conolysin-Mt for native eukaryotic membranes is a novel feature of the peptide compared to other well-characterized cytolytic peptides such as melittin.