The major b-type cytochrome in microsomal membrane preparations from developing endosperm of castor bean (Ricinus communis) was cytochrome b5. Cytochrome P-450 was also present. The microsomal membranes had delta 12-hydroxylase activity and catalysed the NAD(P)H-dependent hydroxylation of oleate to yield ricinoleic acid. CO had no effect on the hydroxylase activity. Rabbit polyclonal antibodies were raised against the hydrophilic cytochrome b5 fragment purified from cauliflower (Brassica oleracea) floret microsomes. The anti-(cytochrome b5) IgG inhibited delta 12-hydroxylase, delta 12-desaturase and cytochrome c reductase activity in the microsomes. The results indicate that electrons from NAD(P)H were transferred to the site of hydroxylation via cytochrome b5 and that cytochrome P-450 was not involved.