The assimilation of (14)CO2 into the C4 acids malate and aspartate by leaves of C3, C4 and C3-C4 intermediate Flaveria species was investigated near the CO2 compensation concentration Γ(*) in order to determine the potential role of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) in reducing photorespiration in the intermediates. Relative to air concentrations of CO2, the proportion of CO2 fixed by PEP carboxylase at Γ(*) increased in all six C3-C4 intermediate species examined. However, F. floridana J.R. Johnston and F. ramosissima Klatt were shown to be markedly less responsive to reduced external CO2, with only about a 1.6-fold enhancement of CO2 assimilation by PEP carboxylase, as compared to a 3.0- to 3.7-fold increase for the other C3-C4 species examined, namely, F. linearis Lag., F. anomala B.L. Robinson, F. chloraefolia A. Gray and F. pubescens Rydb. The C3 species F. pringlei Gandoger and F. cronquistii A.M. Powell exhibited a 1.5- and 2.9-fold increase in labeled malate and aspartate, respectively, at Γ(*). Assimilation of CO2 by PEP carboxylase in the C4 species F. trinervia (Spreng.) C. Mohr, F. australasica Hook., and the C4-like species F. brownii A.M. Powell was relatively insensitive to subatmospheric levels of CO2. The interspecific variation among the intermediate Flaverias may signify that F. floridana and F. ramosissima possess a more C4-like compartmentation of PEP carboxylase and ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) between the mesophyll and bundle-sheath cells. Chasing recently labeled malate and aspartate with (12)CO2 for 5 min at Γ(*) resulted in an apparent turnover of 25% and 30% of the radiocarbon in these C4 acids for F. ramosissima and F. floridana, respectively. No substantial turnover was detected for F. linearis, F. anomala, F. chloraefolia or F. pubescens. With the exception of F. floridana and F. ramosissima, it is unlikely that enhanced CO2 fixation by PEP carboxylase at the CO2 compensation concentration is a major mechanism for reducing photorespiration in the intermediate Flaveria species. Moreover, these findings support previous related (14)CO2-labeling studies at air-levels of CO2 which indicated that F. floridana and F. ramosissima were more C4-like intermediate species. This is further substantiated by the demonstration that F. floridana PEP carboxylase, like the enzyme in C4 plants, undergoes a substantial activation (2.2-fold) upon illuminating dark-adapted green leaves. In contrast, light activation was not observed for the enzyme in F. linearis or F. chloraefolia.