The effect of the adenosine deaminase inhibitor, 2'-deoxycoformycin, on the inhibitory effect of cordycepin on nuclear RNA synthesis was examined in L1210 cells in vitro. The median inhibitory dose for the effect of deoxycoformycin on adenosine deaminase was 4 X 10(-8) M, and 100% inhibition was achieved at 5 X 10(-7) M. Pretreatment of cells for 30 min with 1 X 10(-6) M deoxycoformycin resulted in a reduction of the median inhibitory dose for cordycepin from 2.5 X 10(-4) to 1.8 X 10(-5) M, as assessed by the measurement of [3H]uridine incorporation into total RNA. Measurement of the synthesis of nuclear ribosomal RNA, nonpolyadenylic acid heterogeneous RNA, and polyadenylic acid heterogeneous RNA revealed potentiation by 2'-deoxycoformycin of the inhibitory effect of cordycepin on all species of RNA, as well as on polyadenylic acid synthesis. No differences were noted in the size of nuclear polyadenylic acid obtained from cells treated with cordycepin in either the presence or the absence of the adenosine deaminase inhibitor. These results suggest that the potentiation by 2'-deoxycoformycin of the cytotoxic and antitumor effects of cordycepin on L1210 cells in vivo is related to inhibition of nuclear RNA synthesis.