The effect of recombinant interferon-α2 (IFN-α2) (50 U/ml) on the cell cycle, nucleotide metabolism, and protein and nucleic acid synthesis was studied in human B-lymphoblastoid (Daudi) cells. Cell cycle analysis showed that IFN treatment resulted in G0/G1 arrest (69%) as compared to control cells (42% at G0/G1). IFN inhibited the incorporation of radioactive thymidine and uridine into DNA and RNA, respectively, but had only slight effect on incorporation of radioactive threonine, leucine, or valine into proteins. IFN inhibited ribonucleotide biosynthesis by de novo and salvage pathways and decreased level of the P-ribose-PP. Both pathways of deoxyribonucleotide biosynthesis, ribonucleotide reduction and deoxyribonucleoside salvage, were also markedly inhibited by IFN., In contrast, ribonucleotide catabolism was significantly increased in the presence of IFN. No changes in ribonucleotide interconversion were found. Intracellular concentrations of both ribonucleotides and deoxyribonucleotides were markedly diminished by IFN. These results suggest that inhibition of both ribonucleotide and deoxyribonucleotide biosynthesis, together with increased rate of nucleotide catabolism, may significantly decrease intracellular nucleotide availability. Decrease of the supply of nucleic acid precursors, as well as limitation of nucleotides for energy metabolism and other processes, may result in the inhibition of cell multiplications.