In research to date, regulation of the pyrimidine biosynthetic pathway at the level of gene expression has not been shown for wild type Pseudomonas aeruginosa. No repression was observed when uracil was added to the growth medium nor was any derepression seen when Pyr(-) auxotrophs were limited for pyrimidines. Here we show that the addition of uracil to Pseudomonas minimal medium influenced the synthesis of pyrimidine enzymes, while starvation of a pyrimidine knockout mutant (pyrD) elicited derepression of the pyrimidine enzymes. Moreover, the inclusion of orotate in the growth medium induced the synthesis of dihydroorotase in both wild type and mutant. These results suggest that the pyrimidine pathway in P. aeruginosa is regulated at the level of enzyme synthesis in a manner similar to a number of other Pseudomonas species.