The inhibition of ribosomal RNA (rRNA) maturation by 5-fluorouridine (FUrd) in Novikoff hepatoma cells appears to depend upon the incorporation of the analog into the 45 S rRNA precursor. Precursor synthesized in the presence of FUrd is not processed into mature rRNA, but precursor synthesized in the absence of the analog is processed normally after the addition of the drug. The effect of FUrd on rRNA maturation is concentration dependent. At a concentration of 1 X 10(-4)M, the analog completely inhibits the formation of mature 18 S and 28 S rRNA; while at a concentration of 1 X 10(-7)M, the analog has no significant effect on rRNA maturation. These results suggest that some minimum degree of analog substitution is necessary to inhibit the maturation process. In addition to its inhibition of maturation, FUrd also inhibits the transcription of 45 S rRNA precursor. However, this effect of the drug is less complete and more time dependent that the effect on maturation. The inhibition of rRNA maturation by FUrd persists after removal of the analog from the culture medium. Cells that had been exposed to FUrd for 2 hr were unable to process 45 S rRNA precursor 20 hr after removal of the drug from the medium.