Cholesterol is the main but not the only sterol in cell membranes of higher eukaryotes. Currently, there is an increasing interest toward structurally different sterols, because their membrane partitioning, trafficking, and metabolic properties may differ considerably from those of cholesterol. There is also growing information on specific sterol-protein interactions and their functional consequences, as exemplified by NPC proteins and select ABC-transporters. Several aspects of sterol trafficking and homeostasis are conserved between eukaryotes, and novel, unanticipated findings in this area have recently been made, particularly from genetic screens in yeast. This includes a novel, reversible modification of the sterol structure that affects the choice of transport route.