Incorporation of tritium from thymidine-methyl-H3 (TMH3) in the Laredo strain of Entamoeba histolytica grown at room temperature in the CLG medium, as in the K9 strain grown at 37 C, occurs in both nucleus and cytoplasm. Most extensive cytoplasmic activity is detected during periods of most pronounced growth. Nuclear activity is generally evenly dispersed, but in some instances it is restricted to portions of the peripheral chromatin. The cytoplasmic label is shown to be due in part or wholly to ingestion of DNA of the nonmultiplying protoplasts of the associate cell (Bacteroides) used to support growth of amebae in CLG medium. This cytoplasmic label is unstable and at least some of it serves as precursor material for nuclear DNA of amebae. Experiments done with appropriate RNase and DNAse digestions, and unlabeled carrier compounds thymidine, uridine, thymidylic acid and uridylic acid, indicate that TMH' is highly specific for DNA in this cell system and that the thymidine kinase salvage pathway is probably involved in TMH: utilization. The efficacy of studying patterns of DNA biosynthesis in Entamoeba histolytica by autoradiography using H3-thymidine (H3T) has recently been reported (Albach, Shaffer, and Watson, 1966). These studies described the extent of H3T utilization over the growth cycle of the K9 strain of E. histolytica grown at 37 C in the CLG medium with penicillin-inhibited Bacteroides as the associate organism. The uptake of tritium from H3T was found in a DNasesensitive molecule in both nucleus and cytoplasm of the amebae. Most of the cytoplasmic activity, which was very extensive during periods of pronounced growth, was found to be relatively unstable. It was suggested that the source of DNase-sensitive cytoplasmic activity may be partly or entirely due to ingestion of prelabeled bacterial DNA since tritium from H3-thymidine was also incorporated into DNasesensitive material by the nonmultiplying associate organism. It was thus considered possible that the nuclear activity might be derived from the cytoplasmic label. The purpose of this communication is to extend the observations on the utilization of H3T to the Laredo strain of E. histolytica grown at room temperature in the CLG medium. A preliminary report of these observations has been published (Albach and Shaffer, 1966). Further studies done with the K9 strain, which will provide additional evidence to support the hypothesis that much of the cytoplasmic activity is Received for publication 21 July 1967. * Supported by Grant AI-08004-01 from the NIAID. apparently derived from ingested bacterial DNA which is then used for nuclear DNA synthesis will also be included. MATERIALS AND METHODS Entamoeba histolytica was propagated routinely in the CLG (cysteine-lactalbumin-glucose) medium with the penicillin-inhibited Bacteroides sp. as the associate organism (McDade and Shaffer, 1959). Two strains were used, the K9 strain which was incubated at 37 C and transferred three times weekly, and the Laredo strain which was grown at room temperature and transferred once weekly. Incubation of amebae with exogenous thymidine-methyl-H3 (TMH3) and with unlabeled Bacteroides sp. Cultures of E. histolytica were incubated for varying periods of time with various amounts of TMH3 (Schwarz BioResearch, sp. act. 6 C/mM). In some experiments, unlabeled carrier compounds, thymidine (TdR), thymidylic acid (TMP), uridine (UR), uridylic acid (UMP), obtained from Nutritional Biochemicals Corporation, were added to the medium to yield a final concentration of 0.5 mg/ml. This concentration (ca. 2mM) was approximately 500 to 1,000 times that of the labeled nucleoside. Since it has been demonstrated that thymidine at concentrations above 0.02 mxt may inhibit DNA synthesis and cell growth in some cells (Whittle, 1966; Yang et al., 1966), preliminary experiments were done with all carrier compounds to assure that, at the concentration range used, no such inhibition of cell proliferation occurred. The growth responses of amebae propagated with or without carrier compounds were essentially the same. After the prescribed incubation period in the radioactive medium, the amebae were harvested and washed free of the labeled medium and autoradiographs prepared immediately, or the cells were resuspended in unlabeled medium for a predetermined period of time and then autoradiographs prepared.