We investigated the presence of carbonic anhydrase in root and hypocotyl of etiolated soybean using enzymatic, histochemical, immunohistochemical and in situ hybridization approaches. In parallel, we used in situ hybridization and immunolocalization to determine the expression pattern and localization of phosphoenolpyruvate carboxylase. Their co-localization in the root tip as well as in the central cylinder, suggests that a large fraction of the CO2 may be re-introduced into C4 compounds. GmPK3 expression, coding for a cytoplasmic isoform of pyruvate kinase, was detected in all different root cell types, suggesting that both phosphoenolpyruvate-utilizing enzymes are involved in phosphoenolpyruvate metabolism in etiolated soybean roots; a case indicative of the necessary flexibility plant metabolism has to adopt in order to compensate various physiological conditions.