The effect of drought stress (DS) on photosynthesis and photosynthesis-related enzyme activities was investigated in F. pringlei (C-3), F. floridana (C-3-C-4), F. brownii (C-4-like), and F trinervia (C-4) species. Stomatal closure was observed in all species, probably being the main cause for the decline in photosynthesis in the C3 species under ambient conditions. In vitro ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) and stromal fructose 1,6-bisphosphatase (sFBP) activities were sufficient to interpret the net photosynthetic rates (P-N), but, from the decreases in P-N values under high CO2 (Ca = 700 mu mol mol(-1)) it is concluded that a decrease in the in vivo rate of the RuBPCO reaction may be an additional limiting factor under DS in the C-3 species. The observed decline in the photosynthesis capacity of the C-3-C-4 species is suggested to be associated both to in vivo decreases of RuBPCO activity and of the RuBP regeneration rate. The decline of the maximum P-N observed in the C-4-like species under DS was probably attributed to a decrease in maximum RuBPCO activity and/or to decrease of enzyme substrate (RuBP or PEP) regeneration rates. In the C-4 species, the decline of both in vivo photosynthesis and photosynthetic capacity could be due to in vivo inhibition of the phosphoenolpyruvate carboxylase (PEPC) by a twofold increase of the malate concentration observed in mesophyll cell extracts from DS plants.