P-Glycoprotein (Pgp) (also known as multidrug-resistance protein) contains two nucleotide binding sites, both of which are catalytic ATPase sites. The covalent reagent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) reacts in catalytic sites, and full inactivation of ATPase activity occurs at a reaction stoichiometry of 1 mol of NBD-Cl/mol of Pgp. We show that, at reaction stoichiometry of < or = 1 mol/mol, both nucleotide sites become labeled in relatively nonselective fashion. There is therefore strong interaction between the two nucleotide sites because (a) reaction of one site with NBD-Cl severely impedes reaction of reagent with the other site, and (b) reaction of one site inhibits steady-state ATPase, i.e. both sites are inhibited. Vanadate-trapping experiments revealed that when one nucleotide site was reacted with NBD-Cl, not even a single ATPase turnover event could occur in the other, intact, nucleotide site. The data demonstrate therefore that catalytic cooperativity between the two nucleotide sites in Pgp is extremely strong and mandatory for catalysis.