The multidrug resistance proteins P-glycoprotein (Pgp) and MRPI are drug-efflux pumps, In this study, we compared the nucleotide triphosphatase activities of the isolated N-terminal nucleotide binding domains (NBD1) of Pgp and MRPI, and explored the potential role of the phosphorylation target domain of Pgp on the regulation of Pgp NBD1 ATPase activity. We found that: (1) the NBD1s of Pgp and MRPI have ATPase and GTPase activities, (2) the K(m)s of Pgp NBD1 for ATP and GTP hydrolysis are identical, while the K-m of MRPI NBD1 for ATP is lower than that for GTP, and (3) (p)hosphorylation of MLD by PKA or PKC produces a marginal increase of V-max for ATP hydrolysis, without affecting the affinity for ATP. These results show efficient GTP hydrolysis by the NBD1s of Pgp and MRPI, and a minor role of phosphorylation in the control of Pgp NBD1 ATPase activity. (C) 2002 Elsevier Science (USA). All rights reserved.