Human multidrug resistance protein 4 (MRP4) has recently been determined to confer resistance to the antiviral purine analog 9-(2-phosphonylmethoxyethyl)adenine and methotrexate. However, neither its substrate selectivity nor physiological functions have been determined. Here we report the results of investigations of the in vitro transport properties of MRP4 using membrane vesicles prepared from insect cells infected with MRP4 baculovirus. It is shown that expression of MRP4 is specifically associated with the MgATP-dependent transport of cGMP, cAMP and estradiol 17--D-glucuronide (E₂17G). cGMP, cAMP and E₂17G are transported with K_m and V_max values of 9.7 ± 2.3 [mu]M and 2.0 ± 0.3 pmol/mg/min, 44.5 ± 5.8 [mu]M and 4.1 ± 0.4 pmol/mg/min and 30.3 ± 6.2 [mu]M and 102 ± 16 pmol/mg/min, respectively. Consistent with its ability to transport cyclic nucleotides, it is demonstrated that the MRP4 drug resistance profile extends to 6-mercaptopurine and 6-thioguanine, two anticancer purine analogs that are converted in the cell to nucleotide analogs. On the basis of its capacity to transport cyclic nucleotides and E₂17G it is concluded that MRP4 may influence diverse cellular processes regulated by cGMP and cAMP, and that its substrate range is distinct from that of any other characterized MRP family member.