Anthraquinones (AQs) are compounds with a wide variety of interesting properties, which could be obtained by Rubia tinctorum suspension cultures. The present paper reports the effect of proline and aminoindan-2-phosphonic acid (AIP) on AQs production. The addition of proline (0.25 mM) or AIP (100 mu M) enhance, up to 50% the AQ biosynthesis in R. tinctorum suspension culture. This increase is similar to those obtained by elicitation. Phenylalanine ammonia liase (PAL) activity was higher in presence of proline but no change in phenolic acid contents was found. Glucose-6-phosphate dehydrogenase (G6PDH) activity was not affected in cell cultures treated with proline. Addition of AIP produced PAL inhibition and lower levels of phenolic acid contents. These results show that it is possible to increase the AQs level in R. tinctorum suspension culture by metabolic manipulation and by inhibition of the key enzymes of the metabolic pathways that compete with AQs for a common substrate. (c) 2007 Elsevier Inc. All rights reserved.