Two transposon-induced pyrimidine auxotrophic mutants of Pseudomonas cepacia were analyzed. The enzyme analysis of the pyrimidine biosynthetic pathway within P. cepacia was conducted to biochemically characterize these mutants. Both mutants were found to have a defect in dihydroorotase dehydrogenase, the enzyme that converts dihydroorotic acid to orotic acid. We also found that in P. cepacia cytidine 5'-triphosphate (CTP) inhibited the activity of aspartate transcarbamylase.