Oleate Delta(12)-hydroxylase activity was measured in extracts of developing castor bean seeds. Most of the hydroxylase activity is associated with microsomes. However, when microsomes are washed, the activity is completely lost. Some (50%) of the activity can be restored by addition of the 100,000g supernatant to the washed microsomes. Supernatant extracts (100,000g) of developing safflower seeds are able to restore all (100%) of the hydroxylase activity to the washed castor bean microsomes. In addition, purified mammalian catalase can restore some (25%) of the activity to the microsomes but is not as effective as either castor bean or safflower 100,000g supernatants. The K(m) of the hydroxylase for oxygen is 4 micromolar. Inasmuch as the activity was not inhibited by high concentrations of either carbon monoxide or cyanide, neither the involvement of cytochrome P450 nor other cytochrome systems is suggested. The enzyme system was not saturated by oleoyl-CoA, even at concentrations as high as 200 micromolar. When [(14)C]oleoyl-CoA is supplied as a substrate, the acyl component is rapidly transferred to phosphatidylcholine (PC). Hydroxylation may occur on PC or on a lipid which receives its acyl component from PC. However, exogeneously added 2-[1-(14)C]oleoyl-PC was hydroxylated at a much lower rate than was [1-(14)C]oleoyl-CoA added as the primary substrate.