Chalcone synthase activity was found in enzyme preparations from spinach. In homogenates of young leaves two different activities of the enzyme could be separated by DEAE-ion exchange chromatography and chromatofocusing. Both activities formed naringenin with [2-14C] malonyl- CoA and 4-coumaroyl-CoA as substrates. They exhibited only slight differences in substrate specificity. For both activities 4-coumaroyl-CoA proved to be the most suitable substrate at both pH 6.8 and 8.0. Eriodictyol and homoeriodictyol formation from caffeoyl-CoA and feruloyl-CoA, respectively, only occured at pH 6.8. The formation of naringenin by the two activities was maximal at pH 7.5-8.0 and dependent upon the DTE-concentration in the assay mixture.