Unique mixtures of pheromone components are commonly identified in insects, and have been shown to increase attractiveness towards conspecifics when reconstructed at the natural ratio released by the signaler. In previous field studies of pheromones that attract female sea lamprey (Petromyzon marinus, L.), putative components of the male-released mating pheromone included the newly described bile alcohol 3,12-diketo-4,6-petromyzonene-24-sulfate (DkPES) and the well characterized 3-keto petromyzonol sulfate (3kPZS). Here, we show chemical evidence that unequivocally confirms the elucidated structure of DkPES, electrophysiological evidence that each component is independently detected by the olfactory epithelium, and behavioral evidence that mature female sea lamprey prefer artificial nests activated with a mixture that reconstructs the male-released component ratio of 30:1 (3kPZS:DkPES, molar:molar). In addition, we characterize search behavior (sinuosity of swim paths) of females approaching ratio treatment sources. These results suggest unique pheromone ratios may underlie reproductive isolating mechanisms in vertebrates, as well as provide utility in pheromone-integrated control of invasive sea lamprey in the Great Lakes.