The question of whether bullhead catfish can discriminate binary mixtures of amino acids from the individual components of the mixture was investigated. Two groups of catfish were conditioned to different binary mixtures of L-norvaline (NVAL) and L-leucine (LEU). The concentrations of the amino acids in the conditioned mixtures were adjusted so that in different mixtures either NVAL or LEU was the more stimulatory component. Bullhead catfish were unable to discriminate the more stimulatory components, but were able to discriminate the less stimulatory components and other amino acids from the conditioned mixtures. The third group of bullhead catfish was conditioned to L-proline (PRO) and the responses to different mixtures of PRO and NVAL were subsequently evaluated. Behavioral and electrophysiological (EOG) experiments indicated that the difference in relative stimulatory effectiveness levels between NVAL and PRO is > 30,000 times. For subsequent tests, the concentrations of PRO and NVAL were adjusted to form binary mixtures in which PRO and NVAL, respectively, were the more stimulatory components. Bullhead catfish conditioned to PRO discriminated the mixture if NVAL was the more stimulatory component, but did not discriminate PRO from the mixture if PRO was the more stimulatory component. These results suggest that binary mixtures of amino acids are initially perceived as the more stimulatory components of the mixture.