Expression of a cyclodipeptide synthase gene from Nocardiopsis prasina (CDPS-Np) in Escherichia coli resulted in the formation of cyclo-(l-Tyr-l-Tyr) (1) as the minor and cyclo-(l-Tyr-l-Phe) (2) as the major products. Site-directed mutagenesis revealed a strong influence on product accumulation of the amino acid residues in pocket P1. An 8-fold increase in product formation for 1 and 10-fold for 2 were detected in the double mutant T82V_Y196F compared with the wild type.