4-Monochlorobiphenyl (CB3), mainly an airborne pollutant, undergoes rapid biotransformation to produce hydroxylated metabolites (OH-CB3s). However, up to now, hydroxylation of CB3 has not been studied in living organisms. In order to explore the formation of hydroxylated metabolites of CB3 in whole plants, poplars (Populus deltoides x nigra, DN34) were exposed to CB3 for 10 days. Poplars are a model plant with complete genomic sequence, and they are widely used in phytoremediation. Results showed poplar plants can metabolize CB3 into OH-CB3s. Three monohydroxy metabolites, including 2'-hydroxy-4-chlorobiphenyl (2'OH-CB3), 3'-hydroxy-4-chlorobiphenyl (3'OH-CB3), and 4'-hydroxy-4-chlorobiphenyl (4'OH-CB3), were identified in hydroponic solution and in different parts of the poplar plant. The metabolite 4'OH-CB3 was the major product. In addition, there were two other unknown monohydroxy metabolites of CB3 found in whole poplar plants. Based on their physical and chemical properties, they are likely to be 2-hydroxy-4-chlorobiphenyl (2OH-CB3) and 3-hydroxy-4-chlorobiphenyl (3OH-CB3). Compared to the roots and leaves, the middle portion of the plant (the middle wood and bark) had higher concentrations of 2'OH-CB3, 3'OH-CB3, and 4'OH-CB3, which suggests that these hydroxylated metabolites of CB3 are easily translocated in poplars from roots to shoots. The total masses of 2'OH-CB3, 3'OH-CB3, and 4'OH-CB3 in whole poplar plants were much higher than those in solution, strongly suggesting that it is mainly the poplar plant itself which metabolizes CB3 to OH-CB3s. Finally, the data suggest that the metabolic pathway be via epoxide intermediates.