The A-loop is a recently described conserved region in the NBDs of ABC transporters [Ambudkar, S.V., Kim, I.-W., Xia, D. and Sauna, Z.E. (2006) The A-loop, a novel conserved aromatic acid subdomain upstream of the Walker A motif in ABC transporters, is critical for ATP binding. FEBS Lett. 580, 1049-1055; Kim, I.W., Peng, X.H., Sauna, Z.E., FitzGerald, P.C., Xia, D., Muller, M., Nandigama, K. and Ambudkar, S.V. (2006) The conserved tyrosine residues 401 and 1044 in ATP sites of human P-glycoprotein are critical for ATP binding and hydrolysis: evidence for a conserved subdomain, the A-loop in the ATP-binding cassette. Biochemistry 45, 7605-7616]. In mouse P-glycoprotein (Abcb1a), the aromatic residue of the A-loop in both NBDs is a tyrosine: Y397 in NBD1 and Y1040 in NBD2. Another tyrosine residue (618 in NBD1 and 1263 in NBD2) also appears to lie in proximity to the ATP molecule. We have mutated residues Y397, Y618, Y1040, and Y1263 to tryptophan and analyzed the effect of these substitutions on transport properties, ATP binding, and ATP hydrolysis by Abcb1a (mouse Mdr3). Y618W and Y1263W enzymes had catalytic characteristics similar to WT Abcb1a. On the other hand, Y397W and Y1040W showed impaired transport and greatly reduced ATPase activity, including a approximately 10-fold increase in Km for MgATP. Thus, Y397 and Y1040 play an important role in Abcb1a catalysis.