The mode of action of virustomycin A, a novel 18-membered macrolide, on Trichomonas foetus was investigated. The antibiotic inhibited the biosynthesis of RNA, DNA and protein in the organism. The inhibition of RNA biosynthesis was the most severe. Virustomycin A repressed the incorporation of [3H]uridine into both acid-soluble and insoluble fractions, whereas actinomycin D inhibited the incorporation of [3H]uridine into acid-insoluble fraction alone. Furthermore, it was found that virustomycin A interfered with nucleotide formation from uridine and adenosine but not with their transport to the cells. On the other hand, the antibiotic did not inhibit the activities of uridine kinase and uracil phosphoribosyltransferase in a cell-free extract from the organism. These data suggest that the antibiotic interferes with the formation of phosphate donor(s) (possibly ATP-forming system) of the organism.