Radioiodinated m-iodobenzylguanidine (MIBG), a scintigraphic agent used in the detection of human pheochromocytomas, is thought to utilize the same uptake and retention mechanism(s) as norepinephrine (NE). Using primary cultures from 16 human pheochromocytomas, we compared the uptake of MIBG to that of NE. Two different uptake systems were identified. Both NE and MIBG were taken up by a sodium-dependent system that was characterized by: temperature dependency, high affinity, low capacity, saturability, ouabain sensitivity, and desmethylimipramine sensitivity. However, NE and MIBG were also taken up by a temperature-dependent, sodium-independent, apparently unsaturable system. The sodium-dependent uptake system fulfills many of the criteria for Uptake-one while the sodium-independent uptake system is most likely a passive diffusion process. Competitive inhibition studies demonstrated that NE and MIBG share a common uptake system; a concept consistent with the linear correlation between the rate of uptake of 1.0 microM NE and that of 1.0 microM MIBG (r = 0.942). At low concentrations, both NE and MIBG entered the tumor cells primarily by the sodium-dependent uptake system. Differential expression of the sodium-dependent and sodium-independent uptake systems, between different tumor cells, appears to be responsible for the variations of the kinetic parameters for both NE and MIBG. These studies provide the first direct characterization of a NE uptake mechanism in human pheochromocytoma cells.