Traditionally, proteins belonging to the ATP-binding cassette superfamily have been thought to function exclusively at the plasma membrane (PM) of cells. We have previously shown multidrug resistance-associated protein 1 (MRP1) to reside on the Golgi apparatus of the multidrug resistant (MDR) human leukemic cell line HL-60 (HL-60/ADR); however, neither the prevalence of this abnormal localization nor the functionality of the transporter at the Golgi has been thoroughly addressed. To assess the functionality of MRP1, with respect to its localization in the cell, we transfected MRP1-deficient HeLa cells with an MRP1-enhanced green fluorescent protein (MRP1-EGFP) plasmid. Untreated cells expressed MRP1-EGFP at the PM; however, cells pretreated with monensin caused the transporter to localize on the Golgi apparatus. The MRP1-mediated decline in cytosolic fluorescence of the MRP1 substrate sulforhodamine 101 (SR101) was comparatively evaluated. The rate of decline of SR101 cytosolic fluorescence was found to be of similar magnitude regardless of the localization of MRP1. Additionally, we show that a number of human leukemic cell lines appear to have an inefficient Golgi apparatus to PM secretory pathway that could be responsible for the Golgi localization of MRP1.