OBJECTIVE The invasive sea lamprey (Petromyzon marinus) has devastated the ecosystem of the Laurentian Great Lakes. Application of pheromones to manipulate adult sea lamprey behavior is among the options considered for alternative sea lamprey control techniques. The male sea lamprey sex pheromone is hypothesized to be possess multiple functions through actions of multiple components, some of which have yet to be characterized. Our objective is to isolate and characterize the bioactive components from water conditioned with sexually mature male sea lamprey. METHODS The water conditioned with sexually mature male sea lamprey was extracted by solid phase extraction and concentrated in vacuo. The compounds were isolated by liquid chromatography and elucidated by spectrometry and spectroscopy. Their biological activities were evaluated by electro-olfactogram recordings and two-choice maze behavioral assays. RESULTS Five novel bile salts, petromyzene A and B and petromyzone A-C, have been characterized. Petromyzene A and B featured either a unique, rearranged side chain or a rare cis-11,12-diol on the steroidal B-ring. Petromyzone A-C represented three novel highly oxidized sulfated bile alcohols possessing different hydroxylation, oxidation, and double bond patterns, which exemplify the chemical diversity of bile salts. These five bile salts were potent odorants that stimulated the adult sea lamprey olfactory epithelium in a concentration dependent manner and showed detection thresholds between 10–13 mol·L-1 and 10–11 mol·L-1(paired t-test, P<0.05). Experi?ments in the two-choice maze showed that all isolated compounds induced behavioral responses in ovulated females. CONCLUSION The five novel compounds are likely additional components of phero?mones released by sexually mature male sea lamprey, and may provide useful behavioral manipulation tools to be implemented with the integrated management of the destructive and invasive sea lamprey in the Laurentian Great Lakes.