Bile acids have been implicated as chemical signals in spawning behaviour of lake char (Salvelinus namaycush). In this study, we investigated olfactory responses of lake char to bile acids by using the electro-olfactogram recording. Lake char detected 9 out of 38 bile acids tested at thresholds 0.02-0.5 nM. The most stimulatory included chenodeoxycholic acid, cholic acid, taurochenodeoxycholic acid, taurocholic acid, and taurolithocholic acid 3alpha-sulphate. Structure-activity analysis indicated that substituents in the side chain or hydroxyl sulphation were determinant elements for the recognition of individual bile acid receptors, while the position and orientation of hydroxyls or the type of amidation were important for effective stimulation. Three distinct types of concentration-response relationships were found, representing free, taurine- or glycine-amidated, and 3alpha-sulphated bile acids. Cross-adaptation and binary mixture experiments revealed the presence of multiple olfactory receptors for bile acids. Lake char were also capable of detecting petromyzonol sulphate at 1 nM, possibly via its own receptors. Our study further showed that the olfactory responses to bile acids were independent of those of known odorants including amino acids, prostaglandins and gonadal steroids. We conclude that lake char possess multiple olfactory receptors capable of discriminating bile acids produced and released by conspecifics.