A variety of anti-folate compounds have been tested for their ability to inhibit the growth of Babesia bovis as measured by the incorporation of [3H]hypoxanthine into the parasite's nucleic acids. Inhibitors of folate synthesis (including 7-methylguanosine and several sulpha drugs) were without effect but several structural analogues of folate were toxic. The most potent folate analogues were the lipophilic compounds piritrexim and trimetrexate, each causing 50% inhibition of [3H]hypoxanthine incorporation (IC50) at a concentration of 2.9 nM; other classical anti-folates such as pyrimethamine, methotrexate and trimethoprim were at least 100-fold less effective with IC50 values of 1.2, 0.29 and 0.50 microM, respectively. From these results we conclude that B. bovis does not synthesize folate de novo under cell culture conditions. However, the toxic effects of piritrexim and trimetrexate suggest that dihydrofolate reductase (DHFR) activity is essential for the parasite, most probably because of the role of this enzyme in the synthesis of thymidine nucleotides via thymidylate synthase.