The utilisation (conversion to CO2 and/or glucose) of a series of amino acids by isolated trout hepatocytes was investigated and compared to the utilisation of lactate and palmitate. In fed fish, several amino acids (alanine, serine, asparagine and glycine) and lactate produced CO2 at considerably higher rates than palmitate. During starvation plus exercise, the rate of CO2 production from palmitate increased while that from lactate and most of the amino acids decreased. Gluconeogenesis from amino acids in fed fish was lower than from lactate. Serine and asparagine were the most effective substrates; alanine gave lower rates of incorporation. During prolonged starvation plus exercise, the rates of gluconeogenesis from amino acids increased twofold and, simultaneously, there was a corresponding increase in phosphoenolpyruvate carboxykinase activity in liver. It is concluded that several amino acids (dietary or released from muscle protein) are potentially major oxidative substrates in trout. In addition, amino acids appear to have the capability to maintain supplies of glucose during a period of prolonged starvation and exercise. No evidence could be found to support the contention that alanine is the most important glucogenic amino acid.