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The amount of carbon (psmoles of carbon atoms) drained from the tricarboxylic acid cycle for protein synthesis was compared with pmoles of CO2 released from the cycle at 2-day intervals during the growth of suspension cultures of Paul's Scarlet rose. We concluded that during the period of most rapid protein synthesis (day 0-4) one-sixth as much carbon was drained from the tricarboxylic acid cycle for protein synthesis as was released as CO2. By day 8, one-thirtieth of the amount of carbon released as CO2 was incorporated into protein. Net protein synthesis stopped on day 8, but the evolution of C02/culture continued at its maximum rate until day 10.Similar ratios were calculated based on the recovery of '4C in protein versus CO2 following a 3-hr provision of labeled substrates to 3-day-old cells (age of maximum protein synthesis). Provision of acetate-1-14C and acetate-2-'4C indicated from one-eighth to an equal amount of carbon was incorporated into protein as was released as CO2. When 14C-labeled intermediates of the tricarboxylic acid cycle were provided, the ratio of 14C incorporated into protein versus that evolved in CO2 ranged from 1/0.9 to 1/4.9. Following a critical analysis of the methods used, it was concluded that during periods of rapid protein synthesis, a conservative estimate of the amount of carbon drained from the tricarboxylic acid cycle for protein synthesis was one-fourth of the amount evolved as CO2 from the cycle.Each complete turn of the TCA1 cycle results in the indirect oxidation of one acetate molecule and the release of two CO2 molecules. Thus the number of carbon atoms lost as CO2 matches the number of carbon atoms entering the cycle as acetate, and if this were the only loss the cycle would be self sufficient. However, various intermediates are drained from the cycle and used in amino acid synthesis (5). Carbon removed in this form must be replenished in order for the cycle to continue its operation.The amount of carbon removed from the TCA cycle for amino acid synthesis at different stages of cell development is unknown. In the present investigation, the magnitude of this drainage was studied by comparing the amount of carbon removed from the TCA cycle for protein synthesis with the amount released as CO2 at different stages during the growth of suspension cultures. Two methods were used to make this comparison. In the first approach the ,umoles of carbon entering protein were calculated with a formula based on the net increase in protein measured by the Lowry assay (8), the amino acid composition of protein in rose cells as reported by Dougall 1 Abbreviation: TCA: tricarboxylic acid.(3), and the molecular structure of amino acids derived from TCA cycle intermediates. The ,^moles of CO2 released from the cycle were determined by measuring total CO2 evolution with an IR analyzer and correcting the values for CO2 coming from glycolysis. In the second approach the 14C recovered in protein was compared with that released in CO2 following a 3-hr provision of radioactive TCA cycl...