A temperature-sensitive mutant K12 isolated from an established line of Chinese hamster fibroblasts has been identified as a cell-cycle mutant blocked in G1 (Roscoe et al., '73a,b). While characterizing the phenotype of K12 in more detail, we observed that K12 cells are highly sensitive to the concentration of glucose in the culture medium as well as the temperature of incubation. Although overall cellular metabolism remains intact for at least 24 hours after shifting the cells to the nonpermissive temperature (40.5 degrees C), the incorporation of 3H-leucine into three specific cellular proteins of molecular weights 94, 78, and 58 K daltons is greatly increased. We have analyzed the K12 proteins by two-dimensional, isoelectric focusing (IEF) sodium dodecyl sulfate (SDS) gel electrophoresis and found that the set of proteins which is overproduced by K12 cells at 40.5 degrees C is the same as that produced at 35.0 degrees C in medium depleted of glucose or supplemented with 10 mM glucosamine. In addition, the increased synthesis of these proteins cannot be suppressed by the addition of N-acetylglucosamine to the medium. The relationship of these proteins to the glucose-regulated proteins in the chick embryo fibroblast system (Shiu et al., '77) is discussed.