Acute effects of drugs on incorporation of radioactive precursors into DNA, RNA, and protein of HeLa cells were assayed by means of a sequential isotope technique. The technique involves a 15- to 30-min control preincubation of the cells without drugs, followed by a 30-min experimental incubation with drugs. During the control incubation the medium contains a precursor labeled with 14C; during the experimental period the same precursor is present, now labeled with tritium. The precursors used are 14C- and 3H-thymidine for DNA, 14C- and 3H-uridine for RNA, and 14C- and 3H-leucine for protein. The 3H 14C ratios for groups of monolayers are determined by liquid scintillation techniques. Drugs which change the 3H 14C ratio from that obtained in control monolayers are assumed to have an acute effect. Twenty cytotoxic compounds and two normal nucleosides were tested. Maximal drug concentrations of 10 to 1,000 times the reported IC 50 (that level inhibiting growth of cells in vitro by 50%) usually were employed before excluding an acute effect. Deoxyadenosine, 5-iodo-2-deoxyuridine, cytosine arabinoside, hydroxyurea, and hydroxyurethan inhibited incorporation of thymidine. Puromycin, cycloheximide, and acetoxy-cycloheximide inhibited incorporation of both leucine and thymidine. Actinomycin D inhibited incorporation of uridine and thymidine. Streptonigrin inhibited incorporation of all three precursors. 6-Mercaptopurine, 6-diazo-5-oxo- l-norleucine, urethan, vinblastin, deoxyguanosine, triethylene melamine, and mitomycin C had no significant effect.