Chlorella regularis strain YA-603 was previously found to exhibit a comparatively high growth rate and improved 5-aminolevulinic acid (ALA) productivity when cultured heterotrophically. Although the universal tetrapyrrole precursor ALA is synthesized from glutamate in algae and higher plants, in this study the addition of glutamate to the medium did not enhance ALA production in the heterotrophic culture of C. regularis YA-603. On the other hand, the addition of glycine, which is a precursor of ALA biosynthesis via the Shemin pathway in animal cells and some bacteria, enhanced both the specific growth rate and ALA production. Moreover, when a second glycine addition was made during the culture, the ALA concentration increased by about 1.5 times compared to that obtained with one glycine addition. From these results, it is suggested that the Shemin pathway contributes to ALA production, and that addition of glycine in the heterotrophic culture of C. regularis YA-603 can significantly increase the amount of ALA produced.