Exploration of the role of tryptophan residues in aspartate transcarbamylase (ATCase) was performed by combining site-directed mutagenesis and time-resolved fluorescence measurements. ATCase, an allosteric enzyme of the pyrimidine pathway, is built from three dimeric regulatory subunits and two catalytic trimers. Each catalytic subunit contains two tryptophan residues in position 209 and 284. Two single tryptophan mutants, W209F and W284F were constructed. Analysis by the maximum entropy method of the total fluorescence intensity decays, provides three lifetime classes centered around 0.4 - 0.5, 1.4 - 1.6, and 2.4 - 2.6 ns, respectively, for the wild type enzyme. Analysis of the fluorescence decays permitted attribution of the shorter lifetime to tryptophan in position 284. The isolated catalytic trimers, although devoid of any cooperativity, display very similar fluorescence decays compared to the holoenzymes. In each case, the time-resolved fluorescence anisotropy studies did not evidence any internal flexibility in the nanosecond domain.