Male olfactory cues may guide aggregation on spawning reefs, mate evaluation, and synchronized gamete release in lake trout Salvelinus namaycush, but a lack of information on the source and identity of the cues precludes direct tests of their function. Using a two-channel flume assay, we found female lake trout increased time spent in the channel treated with male-conditioned water, urine, and bile but not synthesized prostaglandin F2α. We suggest future efforts to characterize male olfactory cues focus on urine and postulate that bile acids contribute to its behavioural activity.