Trans-intestinal absorption (TIA) rates for the free amino acids (FAA) alanine, proline, and glutamic acid were measured in two developmental phases of Atlantic herring larvae: Early(14–16 day-post-hatch) and Late (37–39 day-post-hatch). TIA rates were determined by measuring net uptake of a 14C-radiolabeled FAA tracer solution into the body after administration of a single bolus to the digestive tract by tube-feeding. Tracer solutions (concentration range: 0.02 to 100 mM) were administered at < 50% of gut volume (GV) and the response of TIA rate to increasing administered dose (pmol FAA ind− 1) determined. GV was determined as the maximum volume of a tube-fed liquid bolus that could be retained in the digestive tract of a fasted larva without causing any evacuation from the mouth or anus, and was dependent on larval standard length. Our data revealed that alanine, proline and glutamic acid have different TIA rates (pmol min− 1), and results were consistent at both larval stages. At the same administered dose (pmol ind− 1), the TIA rate for glutamic acid was only 8–25% of that of alanine and 15–30% of proline in both larval stages. In Early and Late larvae, the dose-dependent increase in proline and glutamic acid uptake was significantly more rapid than for alanine. Hence, alanine was the most rapidly absorbed FAA at low dose, followed by proline and then glutamic acid, but proline became more rapidly absorbed than alanine at the highest doses, in both stages. In conclusion, the study demonstrated that FAA representing neutral, imino and acidic classes, have different rates of absorption. Across a broad range of concentrations, Atlantic herring larvae have a much lower capacity to deliver glutamic acid from the intestinal lumen to body tissues relative to alanine and proline.