The regulation of hexose transport under glucose-starvation conditions was studied in cultured human skin fibroblasts. Glucose starvation enhanced the transport of 2-DG and 3-0-methyl-D-glucose (3-OMG) but not of L-glucose. Glucose-starvation enhanced transport was inhibited by cytochalasin B (10 microM). The starvation-induced change in 2-DG transport was due to an increase in the Vmax of both the high and low affinity transport sites (2.8- and 2.4-fold, respectively) with no effect on their Kms. The presence of 5.55 mM glucose, fructose, or L-glucose in the medium resulted in transport increases similar to those seen in glucose-starved cells, while the presence of 5.55 mM glucose, mannose, or 3-OMG repressed 2-DG transport. Glucose-starvation enhancement of 2-DG transport was blocked by cycloheximide (20 micrograms/ml) but not by actinomycin D (0.03 microgram/ml) or alpha-amanitin (3.5 microM). Readdition of glucose (5.55 mM) for six hours to glucose-starved cells led to a rapid decrease in hexose transport that could be blocked by cycloheximide but not actinomycin D. Although readdition of 3-OMG to glucose-starved cells had little effect on reversing the transport increases, glucose plus 3-OMG were more effective than glucose alone. Serum containing cultures (10% v/v) of glucose-fed or glucose-starved cells exhibited rapid decreases in 2-DG transport when exposed to glucose-containing serum-free medium. These decreases were prevented by employing glucose-free, serum-free medium. The data indicate that hexose transport regulation in cultured human fibroblasts involves protein synthesis of hexose carriers balanced by interactions of glucose with a regulatory protein(s) and glucose metabolism as they affect the regulation and/or turnover of the carrier molecules.