The transport of histidine and glutamine via system N in cultured hepatocytes was found to be subject to hormonal control. This long-term regulation showed the following characteristics. The transport capacity for histidine and glutamine (system N) increased slowly in response to the combination of dexamethasone and insulin to about 4-fold that of controls after 18-30 h. A similar time course was found for the stimulation of system N (2.5-fold) by dexamethasone and glucagon. In contrast the uptake of alpha-aminoisobutyric acid (system A) was rapidly stimulated 3-fold by dexamethasone and insulin and 5-fold by dexamethasone and glucagon within 3-6 h but decreased towards control rates after 24 h of cultivation in minimal essential medium. Dexamethasone, insulin and glucagon each stimulated glutamine uptake about 2-fold in cultures maintained in W/AB 77 medium, while the combination of dexamethasone with either glucagon or insulin resulted in a 3-4-fold increase. Dexamethasone was most effective at about 0.1 microM. Higher concentrations were less efficient. Insulin reached its optimal effect at concentrations above 1 microM. Kinetic analysis revealed that the increased capacity of glutamine transport in response to hormones was due to an increase in Vmax, while Km was essentially unchanged. The hormone-induced stimulation of system N was prevented by cycloheximide. The induced uptake of glutamine was inhibited by excess amounts of asparagine and histidine but not of alpha-methylaminoisobutyric acid or cysteine. These results clearly differentiate the hormonal regulation of system N from that of system A.