Abstract The inhibition of poly (A) polymerase activity from liver and hepatoma 3924A by several O- n-alky derivatives of rifamycin SV:3-formyloxime was studied. Only the O- n-pentyl (AF/012) and the O- n-octyl (AF/013) analogs were active at the maximum tested concentration of 1 × 10 −4 M. Equivalent concentrations of liver and hepatoma enzymes were inhibited to the same degree by both compounds. The 50 per cent inhibitory concentration (IC 50) for AF/013 and AF/012 was 1.2 × 10 −5M and 7.5 × 10 −5M respectively. Poly (A) polymerase activity was more sensitive to AF/013 if the enzyme was preincubated with the drug before the initiation of the reaction with poly (A) than if the reaction was initiated by the addition of the enzyme. Addition of AF/013 after initiation of the assay led to a rapid inhibition of poly (A) synthesis. The mechanism of inhibition by AF/012 and AF/013 of poly(A) polymerase was of the non-competitive type with respect to ATP.