To investigate the effects of levulinic acid (LA) on extracellular 5-aminolevulinic acid (ALA) accumulation, a heterotrophic culture of Chlorella regularis YA-603 in basal medium containing 0 to 60 mM of LA was carried out. It was found that the extracellular and total ALA concentrations increased, while the chlorophyll (CHL) concentration was the same above initial LA concentrations of 30 mM, with an increasing initial LA concentration up to 50 mM. In the presence of an initial LA concentration of 60 mM, however, the specific growth rate decreased markedly. This suggested that excessive LA exerted a negative effect on ALA production because it also directly affected cellular growth. The relationship between the initial LA and total ALA concentrations mentioned above indicates that LA could promote the ALA-producing activity. Kinetic analysis revealed that an LA concentration of between 30 to 50 mM in the culture broth gave the maximum specific production rate of ALA ((q(ALA))(max)) When LA was added repeatedly to maintain this optimum range of LA concentration, (q(ALA))(max) could be maintained at a relatively high level for a longer period, and the maximum concentration of ALA reached 3.86 mM.