The anthranoid skeleton is believed to be formed by octaketide synthase (OKS), a member of the type III polyketide synthase (PKS) superfamily. Recombinant OKSs catalyze stepwise condensation of eight acetyl units to form a linear octaketide intermediate which, however, is incorrectly folded and cyclized to give the shunt products SEK4 and SEK4b. Here we report in vitro formation of the anthranoid scaffold by cell-free extracts from yeast-extract-treated Cassia bicapsularis cell cultures. Unlike field- and in vitro-grown shoots which accumulate anthraquinones, cell cultures mainly contained tetrahydroanthracenes, formation of which was increased 2.5-fold by the addition of yeast extract. The elicitor-stimulated accumulation of tetrahydroanthracenes was preceded by an approx. 35-fold increase in OKS activity. Incubation of cell-free extracts from yeast-extract-treated cell cultures with acetyl-CoA and [2-14C]malonyl-CoA led to formation of torosachrysone (tetrahydroanthracene) and emodin anthrone, beside two yet unidentified products. No product formation occurred in the absence of acetyl-CoA as starter substrate. To confirm the identities of the enzymatic products, cell-free extracts were incubated with acetyl-CoA and [U-13C3]malonyl-CoA and 13C incorporation was analyzed by ESI-MS/MS. Detection of anthranoid biosynthesis in cell-free extracts indicates in vitro cooperation of OKS with a yet unidentified factor or enzyme for octaketide cyclization.