A NUMBER of reports [1, 5-7, 9-121 have included measurements of protein content of established strains of mammalian cells at various stages of growth and under different culture conditions. The conflicting results apparent in these studies are explainable by the fact, as has been demonstrated by Paul [7], that the environment influences to a very great degree the metabolism and composition of cultured cells. Therefore, when such measurements are used as the basis for determinations of growth [6], or for characterizing certain phases (i.e., lag, log, stationary) of growth in vitro [l, 5, g-121, extreme caution must be used in extrapolating any conclusions drawn therefrom, since they may be valid only in relationship to the particular culture conditions employed. In accordance with this concept, the principal objective of this report is to demonstrate that the primary cell cultures employed herein exhibited drastic changes in protein content throughout their proliferation cycle and in directions opposite to that previously reported [5,10,12] for several established cell lines. Cell suspensions were prepared [4] from freshly excised Walker carcinosarcoma 256 and Jensen sarcoma tumors, which were in a rapid growth phase in vivo, and cultured as monolayers in T-60 flasks. Medium was that referred to previously [3], and initial inoculum was 2 x IO5 cells/ml. Prior to stoppering, each culture was gassed for 30 seconds with a mixture of 5 per cent CO,, 20 per cent 0,, 75 per cent N,. Forty-two and 52 T-60 cultures were used for experiments with Walker and Jensen cells, respectively. The protein from aliquots (60.8 x lo6 Walker and 36.7 y lo6 Jensen cells) of the initial inocula was extracted [2], hydrolyzed by refluxing in 6 N HCl for 18 hours, and analyzed for nitrogen content. In both of these extractions and hydrolyses, and subsequent ones, amounts of extraction solutions and 6 N HCl were varied in proportion to numbers of cells. Medium was changed in the \Valker cultures after cells had adhered to the glass surface (5 hours), and protein nitrogen was determined on representative cultures then and at varying time intervals thereafter. Medium was changed in the Jensen cultures, which took longer to adhere to the glass surface, at 22 hours and again at 71 hours. As before, representative cultures were withdrawn for protein analysis at varying time inervals. Protein nitrogen was determined by the ultramicro method of Tompkins and Kirk [13]. From 3-15 analyses were made on each of the hydrolysates, using lo-100 ,~l samples which contained 2-10 ,ug N. Fig. 1 shows the proliferation curves of the tumor cell cultures, plotted as log, of cell numbers per flask vs. time in hours of incubation. With but one medium change at 5 hours in the Walker cultures, the population increased only from 2.8 x lo6