We have isolated and characterized a cDNA, PfAT208, encoding hydroxycinnamoyl-CoA: anthocyanin 3-O-glucoside-6"-O-acyltransferase (3AT) from Perilla frutescens. The identity of the cDNA was established by determination of the reaction products with recombinant enzyme overexpressed in Escherichia coli. The deduced amino acid sequence has a few regions that are conserved in a CoA-dependent acyltransferase family. The recombinant enzyme produced in yeast could utilize cyanidin 3-glucoside and cyanidin 3,5-diglucoside, putative substrates in vivo, as well as other anthocyanins. The inhibitory effects of diethyl pyrocarbonate and N-ethylmaleimide on the recombinant 3AT activities suggest that histidine and cysteine residues are important for their catalytic function. These properties are in common with anthocyanin 5-O-glucoside-6"-O-acyltransferase (5AT). In Northern analysis, a transcript of PfAT208 was detected in the young leaves of perilla red forma. The properties of other cDNAs, gentian GAT106 and petunia PhAT48, isolated during the above cloning procedure are also described.