The production of L-phenylalanine is conventionally carried out by fermentations that use glucose or sucrose as the carbon source. This work reports on the use of glycerol as an inexpensive and abundant sole carbon source for producing L-phenylalanine using the genetically modified bacterium Escherichia coli BL21(DE3). Fermentations were carried out at 37 degrees C, pH 7.4, using a defined medium in a stirred tank bioreactor at various intensities of impeller agitation speeds (300-500 rpm corresponding to 0.97-1.62 m s(-1) impeller tip speed) and aeration rates (2-8 L min(-1), or 1-4 vvm). This highly aerobic fermentation required a good supply of oxygen, but intense agitation (impeller tip speed approximately 1.62 m s(-1)) reduced the biomass and L-phenylalanine productivity, possibly because of shear sensitivity of the recombinant bacterium. Production of L-phenylalanine was apparently strongly associated with growth. Under the best operating conditions (1.30 m s(-1) impeller tip speed, 4 vvm aeration rate), the yield of L-phenylalanine on glycerol was 0.58 g g(-1), or more than twice the best yield attainable on sucrose (0.25 g g(-1)). In the best case, the peak concentration of L-phenylalanine was 5.6 g L(-1), or comparable to values attained in batch fermentations that use glucose or sucrose. The use of glycerol for the commercial production of L-phenylalanine with E. coli BL21(DE3) has the potential to substantially reduce the cost of production compared to sucrose- and glucose-based fermentations.