Arabidopsis seedlings grown for 14 d without phosphate (P) exhibited stunted growth and other visible symptoms associated with P deficiency. RNA contents in shoots decreased nearly 90%, relative to controls. In shoots, expression of Pht1;2, encoding an inducible high-affinity phosphate transporter, increased threefold, compared with controls, and served as a molecular marker for P limitation. Transcript levels for five enzymes (aspartate transcarbamoylase, ATCase, EC 2.1.3.2; carbamoyl phosphate synthetase, CPSase, EC 6.3.5.5); UMP synthase, EC 2.4.1.10, EC 4.1.1.23; uracil phosphoribosyltransferase, UPRTase, EC 2.4.2.9; UMP kinase, EC 2.7.1.14) increased 2-10-fold in response to P starvation in shoots. These enzymes, which utilize phosphorylated intermediates at putative regulated steps in de novo synthesis and salvaging pathways leading to UMP and pyrimidine nucleotide formation, appear to be coordinately regulated, at the level of gene expression. This response may facilitate pyrimidine nucleotide synthesis under P limitation in this plant. Expression of P-dependent and P-independent phosphoribosyl pyrophosphate (PRPP) synthases (PRS2 and PRS3, respectively) which provide PRPP, the phosphoribosyl donor in UMP synthesis via both de novo and salvaging pathways, was differentially regulated in response to P limitation. PRS2 mRNA levels increased twofold in roots and shoots of P-starved plants, while PRS3 was constitutively-expressed. PRS3 may play a novel role in providing PRPP to cellular metabolism under low P availability.