Large neutral amino acids (LNAAs) compete with each other for carrier-mediated transport through the blood-brain barrier into the brain. The relative plasma concentration, expressed as the ratio of each LNAA to the sum of LNAAs, is considered the main regulator of brain LNAA concentrations. In order to investigate the consistency of this assumption throughout a 24-h period, we have compared the relationship of plasma LNAAs to brain LNAAs among groups of rats fed diets containing various amounts of protein (in order to obtain a wide range of plasma LNAA levels) at two different phases of the light/dark cycle (0900 and 2100 hours). The relationship between plasma and brain LNAAs was found to be dependent on both diet and the time of day. Similar plasma amino acid concentrations in the morning and in the evening contrasted with different brain concentrations. Furthermore, previous findings that brain LNAA concentrations are influenced by plasma amino acid concentrations were confirmed.