The enzyme phenylalanine hydroxylase, the substrate phenylalanine, the product of the reaction tyrosine, and the probable in vivo cofactors (6R)-l-erytro-5,6,7,8-tetrahydrobiopterin (H4Bip) and 5,6,7,8-tetrahydropterin (H4Ptr), have been measured during development in Drosophila. The developmental profile of phenylalanine hydroxylase activity shows two peaks. The larger occurs at the time of pupation, coiciding with an important accumulation of tyrosine in the insect. The minor peak appears at the time of adult emergence. The developmental profile of H4Bip shows also two peaks, coinciding with those of maximal phenylalanine hydroxylase activity. However, H4Ptr is only detectable after late pupa, with a small peak at adult emergence. The results of the analyses of the phenylalanine hydroxylase activity in vitro showed that H4Bip is a better cofactor than H4Ptr, based on the difference in Vmax (4–5 times) and in Km for phenylalanine (1.6 times). Moreover, the difference in activity of the enzyme with H4Bip related to that with H4Ptr, increases even more when larvae are reared on phenylalanine-supplemented media (40-fold difference in Vmax). Also, the inhibition of H4Bip synthesis with N-acetylserotonin resulted in an increase of phenylalanine. As in most other organisms studied, our results indicate that H4Bip is by far the preferred natural cofactor for the Drosophila phenylalanine hydroxylase.