The uptake of 3-O-methylglucose by rat thymocytes follows a biphasic time course. 2,4-Dinitrophenol (10-3 M), carbonyl cyanide m-chlorophenylhydrazone (10-5 M) and oligomycin (5 microgram/ml) each reduce ATP levels in rat thymocytes by 85% and bring about 3- to 4-fold stimulation of the slow phase of 3-O-methylglucose uptake. No consistent effect is observed on either the half-time of the rapid phase of uptake or the relative proportions of the two phases of uptake in the presence of these agents. Ca2+ ions do not appear to play a necessary role in the stimulation of transport activity since cells depleted of exchangeable Ca2+ by treatment with the Ca2+-Mg2+ ionophore, A23187, plus [ethylenebis(oxyethylenenitrilo)]tetraacetic acid respond to uncouplers in exactly the same manner as untreated cells. The effect of dinitrophenol on the slow phase of 3-O-methylglucose uptake is reversible after 10 min of incubation. After 60 min however, cells washed free of dinitrophenol and incubated at 37 degrees exhibited an additional acceleration in transport activity. This stimulation of transport is characterized by an increase in the proportion of the rapid phase of uptake with little or no change in its half-time. The results suggest that rat thymocytes regulate their 3-O-methylglucose transport activity in two distinct fashions.