Polyprenylated acylphloroglucinol derivatives, such as xanthones, are natural plant products with interesting pharmacological properties. They are difficult to synthesize chemically. Biotechnological production is desirable but it requires an understanding of the biosynthetic pathways. cDNAs encoding membrane-bound aromatic prenyltransferase (aPT) enzymes from Hypericum sampsonii seedlings (HsPT8px and HsPTpat) and Hypericum calycinum cell cultures (HcPT8px and HcPTpat) were cloned and expressed in Saccharomyces cerevisiae and Nicotiana benthamiana, respectively. Microsomes and chloroplasts were used for functional analysis. The enzymes catalyzed the prenylation of 1,3,6,7-tetrahydroxyxanthone (1367THX) and/or 1,3,6,7-tetrahydroxy-8-prenylxanthone (8PX) and discriminated nine additionally tested acylphloroglucinol derivatives. The transient expression of the two aPT genes preceded the accumulation of the products in elicitor-treated H. calycinum cell cultures. C-terminal yellow fluorescent protein fusions of the two enzymes were localized to the envelope of chloroplasts in N. benthamiana leaves. Based on the kinetic properties of HsPT8px and HsPTpat, the enzymes catalyze sequential rather than parallel addition of two prenyl groups to the carbon atom 8 of 1367THX, yielding gem-diprenylated patulone under loss of aromaticity of the gem-dialkylated ring. Coexpression in yeast significantly increased product formation. The patulone biosynthetic pathway involves multiple subcellular compartments. The aPTs studied here and related enzymes may be promising tools for plant/microbe metabolic pathway engineering.