The enzyme 4-coumarate:CoA ligase (4CL) plays a key role in channelling carbon flow into diverse branch pathways of phenylpropanoid metabolism which serve important functions in plant growth and adaptation to environmental perturbations. Here we report on the cloning of the 4CL gene family from Arabidopsis thaliana and demonstrate that its three members, At4CL1, At4CL2 and At4CL3, encode isozymes with distinct substrate preference and specificities. Expression studies revealed a differential behaviour of the three genes in various plant organs and upon external stimuli such as wounding and UV irradiation or upon challenge with the fungus, Peronospora parasitica. Phylogenetic comparisons indicate that, in angiosperms, 4CL can be classified into two major clusters, class I and class II, with the At4CL1 and At4CL2 isoforms belonging to class I and At4CL3 to class II. Based on their enzymatic properties, expression characteristics and evolutionary relationships, At4CL3 is likely to participate in the biosynthetic pathway leading to flavonoids whereas At4CL1 and At4CL2 are probably involved in lignin formation and in the production of additional phenolic compounds other than flavonoids.