The effect of several rifamycin derivatives on poly(A) synthesis in vitro was tested using purified rat liver mitochondrial poly(A) polymerase assayed with an exogenous primer. When used at a concentration of 300 mug/ml, derivatives AF/013, PR/19, AF/AETP, M/88 and AF/ABDP completely inhibited activity corresponding to 50 mug of enzyme protein. Under similar conditions, derivatives DMAO and AF/MO failed to inhibit enzyme activity. Studies with PR/19 showed that the drug interacted directly with the enzyme molecule and did not affect the enzyme-primer complex formation. The inhibition by the drug could be reversed by increasing the substrate (ATP) concentration. It is concluded that some rifamycin derivatives can specifically inhibit template-independent nucleotide chain elongation reactions.