Novel proline 3-hydroxylase (P3H) and proline 4-hydroxylase (P4H) activities that convert free L-proline to both trans 3- and trans 4-hydroxy-L-proline were detected in protein extracts of the anamorphic fungus Glarea lozoyensis. The enzymatic conversion Of L-proline to trans 3- and trans 4-hydroxy-L-proline was strictly dependent on a-ketoglutarate, ascorbate, and dithiothreitol. Ferrous iron was required for optimal P3H and P4H activity. These substrate and co-factor requirements indicate these enzyme activities belong to the class of 2-oxoglutarate-dependent dioxygenases. Both P3H and P4H were inhibited by zinc and other trace metals. The addition of proline to the fermentation medium resulted in an increase in the specific activity of P4H and a decrease in the synthesis of pneumocandin Co. Additionally, the synthesis of trans 3- and trans 4-hydroxy-L-proline in vivo was affected differently by the proline concentration in the medium. This result suggested that two enzymes may be responsible for the regio- and stereospecific hydroxylation Of L-proline.