beta-Poly(L-malate) (PMLA) production in Physarum polycephalum has been followed by using D-[1-C-13]glucose and (CaCO3)-C-13. Nuclear magnetic resonance studies of PMLA showed that the C-13 label from [1-C-13]glucose was incorporated in the presence of CaCO3 into positions C-3 (-CH2-) and C-4 (-CO-) of the L-malate repeating unit of PMLA. The C-13 label from (CaCO3)-C-13 was incorporated into position C-4 and indicated that not only the endogenous CO2 but also the exogenous CO2 from CaCO3 served significantly as a carbon source for PMLA production. In the absence of CaCO3, the C-13 labeling pattern of PMLA from D-[1-C-13]glucose was almost indistinguishable from that for the natural abundance C-13-NMR spectrum of the polymer. These results indicated that L-malate used for PMLA production is synthesized either via carboxylation of pyruvate and reduction of oxaloacetate in the presence of CaCO3 or via the oxidative tricarboxylic acid (TCA) cycle in the absence of CaCO3. Avidin strongly inhibited the formation of L-malate via carboxylation; the C-13 labeling pattern of PMLA in the presence of CaCO3 was almost identical with that for the natural abundance spectrum when avidin was added, indicating that L-malate utilized for PMLA production was supplied under this condition by the oxidative TCA cycle.