The sea lamprey is an ancient, parasitic fish that invaded the Great Lakes a century ago, where it triggered the collapse of many fisheries. Like many fishes, this species relies on chemical cues to mediate key aspects of its life, including migration and reproduction. Here we report the discovery of a multicomponent steroidal pheromone that is released by stream-dwelling larval lamprey and guides adults to spawning streams. We isolated three compounds with pheromonal activity (in submilligram quantities from 8,000 l of larval holding water) and deduced their structures. The most important compound contains an unprecedented 1-(3-aminopropyl)pyrrolidin-2-one subunit and is related to squalamine, an antibiotic produced by sharks. We verified its structure by chemical synthesis; it attracts adult lamprey at very low (subpicomolar) concentrations. The second component is another new sulfated steroid and the third is petromyzonol sulfate, a known lamprey-specific bile acid derivative. This mixture is the first migratory pheromone identified in a vertebrate and is being investigated for use in lamprey control.