An enzyme involved in the biosynthesis of phytoecdysteroids, the C2‐hydroxylase, has been investigated in spinach, Spinacia oleracea. This enzyme is microsomal and its K_m has been determined using 2‐deoxy‐20‐hydroxyecdysone as substrate (K_m=3.72 µM). It is much more efficient with 2‐deoxy‐20‐hydroxyecdysone than with 2‐deoxyecdysone and, conversely, the C20‐hydroxylase is more active on 2‐deoxyecdysone than on ecdysone. These data support the conclusion that C20‐hydroxylation precedes C2‐hydroxylation. The C2‐hydroxylase is inhibited by high concentrations of 20E. Substrate specificity and subcellular localization of C2‐hydroxylase differ between plants and insects, and these data, as well as those previously reported on other biosynthetic steps, show the great difference between plant and insect ecdysteroid biosynthetic pathways and suggest an independent origin for the pathways in both kingdoms. © 2009 Wiley Periodicals, Inc.