The amounts of copper present in highly purified preparations of L-tryptophan 2,3-dioxygenase from Pseudomonas fluorescens have been shown to be negligible by six different methods of copper determination. It has also been demonstrated that, during the purification, the heme content of enzyme preparations increased in parallel with the specific enzyme activity, whereas that of copper decreased. These results, together with the finding that the inhibitory effects of copper chelators on the enzyme could be attributable to some other action of these chemicals rather than to their chelating properties, indicate that copper is not an essential component of L-tryptophan 2,3-dioxygenase.