To identify potential hormonal pheromones of the round goby (Neogobius melanostomus), a species recently introduced to the Great Lakes, we used electro-olfactogram (EOG) recording to examine olfactory responsiveness to more than 100 steroids and prostaglandins. Neogobius detected free and conjugated 18-, 19and 21-carbon steroids, but did not detect prostaglandins. EOG cross-adaptation, used to determine if Neogobius can discriminate the detected compounds at the sensory level, suggested that the detected steroids act on four classes of olfactory receptor mechanisms named (according to the most potent ligand for each): estrone, 17β-estradiol-3β-glucuronide, etiocholanolone, and dehydroepiandrosterone3-sulfate. Although none of the detected steroids induced reproductive behaviors, exposure to steroids from three of the four receptor classes (estrone, 17β-estradiol-3β-glucuronide, or etiocholanolone) increased ventilation rate in males, whereas only etiocholanolone increased ventilation rate in females. Using the ventilation increase as a behavioral bioassay of steroid detection, behavioral cross-adaptation studies in males demonstrated that steroids discriminated at the sensory level are also discriminated behaviorally. These findings suggest the round goby may use steroids as putative pheromones.