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In the cultivation of barramundi Lates calcarifer, one of the key factors is developing new commercial feeds that are nutritionally adequate and attractive to fish. The sensory quality of feeds can be improved by taste stimulants. The aim of the present study is to examine the taste attractiveness of 21 free L-amino acids and 4 basic taste substances (0.1–0.001 M). The feeding behavior that accompanied the orosensory testing of feed is also studied. Agar pellets flavored with each taste substance were individually offered to barramundi juveniles (5–9 cm, 4–10 g). Citric acid, cysteine, and alanine were palatable; sucrose and 7 amino acids had an aversive taste; sodium chloride, calcium chloride, and 12 amino acids did not influence the consumption of pellets. Taste preferences for amino acids are positively correlated in 6 out of 21 fish species, which confirms high species specificity of taste preferences in fishes. Barramundi often make repeated grasps and rejections of pellets regardless of their palatability, which led to the destruction of pellets in up to 50% of trials. When swallowing, fish retain pellets several times longer, and pellet fragmentation occurs more often, than in the case of final refusal of pellets. The data presented here can be used to improve the cultivation of barramundi.