The rate of label incorporation of various precursors into liver glycogen and exhaled carbon dioxide was investigated in rats 3, 6, 24 and 48 hours after cortisol injection. The glycogen concentration in the liver attained its maximum 24 hours after the hormone injection, whereas the total radioactivity of glycogen from [1-14C] glucose, [1–3-14C] lactate and [1,2-14C] glycerol reached the highest level during 3 to 6 hours and then remained within the subnormal limits 24 hours after the injection. The label incorporation from [1-14C] glycine into liver glycogen reached a maximum 6 to 24 hours after the hormone injection, coinciding in time with the maximum increase of the glycogen concentration in the liver. Hence, amino acids play a more important role in glycogen formation as compared with other precursors; thus they seem to be the main substrates in cortisol-induced gluconeogenesis. Cortisol produced no effect on [1,2-14C] glycerol oxidation, but the hormone increases oxidation of other labelled substrates involved in exhaled carbon dioxide.