Previous studies have demonstrated that the active metabolite of leflunomide, A77 1726 [N-(4-trifluoromethylphenyl-2-cyano-3-hydroxycrotoamide)], is capable of inhibiting the activities of tyrosine kinases and dihydroorotate dehydrogenase (DHO-DHase). In the present study, we define the relative contribution of these activities to the ability of A77 1726 to inhibit proliferation of the murine leukemia cell line LSTRA. A77 1726 inhibited LSTRA cell growth and proliferation (IC50 = 10-30 microM); this inhibition, however, could be reversed by the addition of exogenous uridine, suggesting that the anti-proliferative activity of A77 1726 may be due to inhibition of de novo pyrimidine nucleotide synthesis. Quantitation of nucleotide levels revealed that A77 1726, at an IC50 of about 10 microM, selectively inhibited pyrimidine nucleotide but not purine nucleotide synthesis. In vitro enzyme assays confirmed that A77 1726 directly inhibited the activity of DHO-DHase, the fourth enzyme in the de novo pathway of pyrimidine nucleotide synthesis (IC50 = 220 nM). LSTRA cells overexpress p56lck and have elevated levels of tyrosine phosphorylated intracellular proteins. A77 1726 reduced the intracellular levels of tyrosine phosphorylated proteins with relatively high IC50 values ranging from 50 to 100 microM. A77 1726 also inhibited p56lck activity in LSTRA membrane preparation and immunoprecipitates; the IC50 values for inhibition of immunoprecipitated p56lck autophosphorylation and exogenous substrate histone 2B were 80 and 40 microM, respectively. The anti-tyrosine phosphorylation activity of A77 1726 was not affected by uridine. These studies therefore demonstrate the two activities of A77 1726: inhibition of pyrimidine nucleotide synthesis and interference with tyrosine phosphorylation.