D-Glucose and D-galactose were the starting materials of L-ascorbic acid biosynthesis in Euglena gracilis as evidenced by feeding experiments of unlabeled and labeled sugars, but D-glucose was the more effective precursor. The addition of various acid derivatives of D-glucose and D-galactose, with the exception of D-glucono-δ-lactone, considerably augmented L-ascorbic acid formation. D-Galacturonic acid and L-galactono-γ-lactone showed greater effects than did D-glucurono-γ-lactone and L-gulono-γ-lactone. The results of isotopic dilution experiments also showed the preference for the galacto-configuration. Fed U-¹⁴C-D-glucose was transformed into labeled D-galacturonic acid to a greater extent than into labeled D-glucuronic acid, and added D-galacturonic acid only caused extensive accumulation of labeled D-galacturonic acid. These results together show that the pathway involving D-galacturonic acid and L-galactono-γ-lactone is the major one, the one involving D-glucuronic acid L-gulono-γ-lactone being the minor one. A likely pathway for L-ascorbic acid biosynthesis in Euglena is proposed in the Scheme, which thus involves uronic acid intermediates and configurational inversion.