Blue and violet flowers generally contain derivatives of delphinidin; red and pink flowers generally contain derivatives of cyanidin or pelargonidin. Differences in hydroxylation patterns of these three major classes of anthocyanidins are controlled by the cytochrome P450 enzymes flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase. Here we report on the isolation of complementary DNA clones of two different flavonoid 3',5'-hydroxylase genes that are expressed in petunia flowers. Restriction-fragment length polymorphism mapping and complementation of mutant petunia lines showed that the flavonoid 3',5'-hydroxylase genes correspond to the genetic loci Hf1 and Hf2.