The pore forming capacity of Sticholysin II (StnII; isolated from Stichodactyla helianthus) in bilayer membranescontaining 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), palmitoylsphingomyelin (PSM) and eithercholesterol or palmitoyl ceramide (PCer) has been examined. The aim of the study was to elucidate howthe presence of differently ordered PSM domains affected StnII oligomerization and pore formation. Cholesterolis known to enhance pore formation by StnII, and our results confirmed this and provide kinetic information forthe process. The effect of cholesterol on bilayer permeabilization kinetics was concentration-dependent. In theconcentration regime used (2.5–10 nmol cholesterol in POPC:PSM 80:20 by nmol), cholesterol also increasedthe acyl chain order in the fluid PSM domain and thus decreased bilayer fluidity, suggesting that fluidity per sewas not responsible for cholesterol's effect. Addition of PCer (2.5–10 nmol) to the POPC:PSM (80:20 by nmol) bilayersattenuated StnII-induced pore formation, again in a concentration-dependent fashion. This addition alsoled to the formation of a PCer-rich gel phase. Addition of cholesterol to PCer-containingmembranes could partiallyreduce the inhibitory effect of PCer on StnII pore formation.We conclude that the physical state of PSM(as influencedby either cholesterol or PCer) affected StnII binding and pore formation under the conditions examined.