The rate of "4C-leucine and 3H-uracil incorporation by tobacco cells (Nicotiana tabaccum var. Samsun N.N.) in suspension culture was simultaneously decreased by the addition of kinetin at concentrations above 2.5 X 10-5 M.Ribosomal RNA was the first RNA species affected by kinetin. The purine derivatives, adenine and N6-methylaminopurine, which exhibit low cytokinin activity overcame the inhibitory effects of kinetin. However, purine derivatives without cytokinin activity, guanine, N6. -dimethylaminopurine, and 2-aminopurine, did not relieve kinetin inhibition.Since the early studies of cytokinins, it has been shown that in addition to their stimulatory effect on the growth of plant tissues they exhibited some inhibitory effects. Skoog and Miller (20) have shown that tobacco stem segments which were plated on medium containing 0.5 mg/l of kinetin formed only few buds, and that these buds were retarded in their growth. The growth of the basal callus was also inhibited by kinetin. Other reports demonstrate the inhibitory effect of kinetin on buds, roots, and shoot formation in intact plant and plant tissues (4,5,8,23).In general, substances with cytokinin activity stimulated the growth of tobacco callus tissue, but high concentrations of them inhibited the growth of the same tissue (10,19). Cytokinins stimulate the synthesis of RNA and protein in plant tissue (16,21,22), in isolated nuclei (3, 17), and isolated mitochondria (1). In this paper we studied the kinetics of the inhibition by high concentration of kinetin, of 3H-uracil, and '4C-leucine incorporation by tobacco cells in suspension culture. A preliminary report has been published elsewhere (14 3H-Uracil and 14C-Leucine Incorporation. The rate of incorporation of 3H-uracil and '4C-leucine was determined in suspensions of single cells and very small cell clusters. These suspensions were obtained by precipitating the large clusters and collecting the supernatant of the stock cultures. Unless otherwise mentioned in the text, radioactive precursors, hormones, or drugs were added to the suspensions at zero time. After varying periods of time, aliquots of the suspensions were transferred into trichloroacetic acid (10% final concentration) for 20 min. The cells were precipitated on Whatman fiberglass filters and washed with 5% trichloroacetic acid. Radioactivity was measured in a Packard Tricarb liquid scintillation spectrometer. Most of the 3H-and '4C radioactivity could be removed from the filters by treatment with ribonuclease A (Worthington) and pronase (Calbiochem), respectively.Labeling ofRNA with 32P, Extraction and Separation on Methylated Albumin Kieselguhr Columns. Four days prior to the labeling experiments, the cultures were transferred into a phosphate-poor medium (one-eighth of its concentration in the growth medium). Following the incubation of the cultures with 1 mc of carrier-free 32PO43-(Amersham) the tissue (10 g fresh wt) was collected on a precooled Buchner funnel and washed with an excess of ice cold water. The tissue was then froze...