A novel phenylacetyl-CoA ligase gene, designated phlB, was cloned and identified from the penicillin producing strain Penicillium chrysogenum based on subtractive suppression hybridization approach. The phlB gene contains a 1686-bp open-reading frame and encodes a protein of approximately 62.6 kDa. The deduced amino acid sequence shows about 35% identity to the characterized P. chrysogenum phenylacetyl-CoA ligase Phl and has a peroxisomal targeting signal on its C-terminal. Recombinant PhlB protein was overexpressed in Escherichia coli and purified by nickel affinity chromatography. Enzymatic assay confirmed that recombinant PhlB can catalyze the reaction of phenylacetic acid (PAA) with CoA to yield phenylacetyl-CoA. The expression level of phlB in the penicillin producing medium supplemented with PAA, the side chain precursor of penicillin G, was about 2.5-fold higher than that in medium without PAA. The study suggested that PhlB might participate in the activation of PAA during penicillin biosynthesis in P. chrysogenum.