We have previously shown that GTP can replace ATP as an energy source to support vinblastine transport by the multidrug transporter P-glycoprotein (Pgp) in plasma membrane vesicles isolated from the multidrug resistant cell line KB-V1 [Lelong et al. (1992) FEBS Lett. 304, 256-260]. Like [gamma-32P]ATP, [gamma-32P]GTP was also able to phosphorylate Pgp in vitro. Unlabeled GTP enhanced the phosphorylation of the transporter by [gamma-32P]ATP, whereas unlabeled ATP inhibited incorporation of label. While phosphorylation by [gamma-32P]ATP was Mg(2+)-dependent, the enhanced phosphorylation of Pgp by GTP was supported by Mg2+ or Mn2+ and to a lesser extent, Ca2+. Specific inhibitors of cAMP-dependent protein kinase, protein kinase C and cGMP-dependent protein kinase, did not affect phosphorylation. The phosphoprotein phosphatase inhibitor okadaic acid slightly enhanced phosphorylation, and vanadate more dramatically increased phosphorylation of the transporter. Tryptic maps of Pgp phosphorylated peptides indicate that addition of GTP altered the relative labeling of phosphopeptides. These results suggest that the overall phosphorylation of Pgp in vitro is determined by several different protein kinases and phosphatases, at least one of which may be GTP-regulated.