The anaerobic utilization of L-ascorbate by gene products of the ula regulon in Escherichia coli has been widely documented. Under aerobic conditions, we have shown that this metabolism is only functional in the presence of casein acid hydrolysate. Transcriptional fusions and proteomic analysis indicated that both the ula regulon and the yiaK-S operon are required for the aerobic utilization of this compound. The aerobic dissimilation of l-ascorbate shares the function of three paralogous proteins, UlaD/YiaQ, UlaE/YiaR and UlaF/YiaS, which encode a decarboxylase, a 3-epimerase and a 4-epimerase, respectively. In contrast, l-ascorbate enters the cells through the ula-encoded phosphotransferase transport system, but it is not carried by the yiaMNO-encoded ABC transporter. Proteomic analysis also indicated enhanced expression of the alkyl hydroperoxide reductase encoded by the ahpC gene, suggesting a response to oxidative stress generated during the aerobic metabolism of l-ascorbate. Control of ahpC expression by the OxyR global regulator in response to l-ascorbate concentration is consistent with the formation of hydrogen peroxide under our experimental conditions. The presence of certain amino acids such as proline, threonine or glutamine in the culture medium allowed aerobic l-ascorbate utilization by Escherichia coli cells. This effect could be explained by the ability of these amino acids to allow yiaK-S operon induction by l-ascorbate, thus increasing the metabolic flux of l-ascorbate dissimilation. Alternatively, these amino acids may slow the rate of L-ascorbate oxidation.