Alkynes are mechanism-based inhibitors of several bacterial monooxygenases, including the soluble methane monooxygenase (sMMO) of Methylococcus capsulatus and the toluene o-monooxygenase (TOM) of Burkholderia cepacia G4. In this paper, we investigated the inhibition of the phenol hydroxylase of Pseudomonas sp. CF600 by the alkyne phenylacetylene. Growth of CF600 on phenol and phenol hydroxylase activity were inhibited by phenylacetylene concentrations greater than 1.0 mM. Unlike other alkynes, which irreversibly inhibit a number of monooxygenases, inhibition of phenol hydroxylase by phenylacetylene was reversible, as demonstrated by the ability of washed cells to regain phenol hydroxylase activity. Additionally, phenylacetylene was metabolized by phenol-grown cells, yielding a yellow meta-ring fission product which absorbed light maximally at 412 nm. Phenol-grown CF600 transformed phenylacetylene to hydroxyphenylacetylene and 2-hydroxy-6-oxo-octa-2,4-dien-7-ynoic acid as detected by gas chromatography-mass spectroscopy and high-performance liquid chromatography (HPLC), respectively, while neither a derivative of CF600 with a non-functional phenol hydroxylase nor wild-type CF600 grown on acetate transformed phenylacetylene. These results demonstrate that the phenol hydroxylase of CF600 has broader substrate specificity than previously reported. They also suggest that phenylacetylene acts as a competitive inhibitor rather than as a mechanism-based inhibitor of this phenol hydroxylase.