The distribution of the two chalcone synthases in leaves ofSpinacia oleracea L. was studied at both the tissue and the subcellular level using immunofluorescence and immunogold techniques. Neither technique differentiated between the two enzyme forms. The chalcone synthases are located in the upper and the lower epidermis and to a minor extent in the subepidermal layers. Traces of the two enzyme forms may be present in the residual mesophyll. This distribution is independent of leaf age. A similar distribution of chalcone synthase among tissues was observed in parsley, pea, and bean. Chalcone synthase is also present in guard cells. The spinach chalcone synthases are cytosolic enzymes, and are not associated with tonoplast or endoplasmic reticulum. A small fraction of the chalcone synthases is located in the stroma of the chloroplasts.