The amino acid sequence (L67) intervening between the M6 and M7 transmembrane segments of the Ca(2+) transport ATPase was subjected to mutational analysis. Mutation of Pro(820) to Ala interferes with protein expression even though transcription occurs at normal levels. Single mutations of Lys(819) or Arg(822) to Ala, Phe, or Glu allow good expression, but produce strong inhibition of ATPase activity. The main defect produced by these mutations is strong interference with enzyme phosphorylation by ATP in the presence of Ca(2+), and also by P(i) in the absence of Ca(2+). The Lys(819) and Arg(822) mutants undergo slight and moderate reduction of Ca(2+) binding affinity, respectively. Reduction of overall steady state ATPase velocity is then due to inhibition of phosphorylated intermediate formation. On the other hand, a cluster of conservative mutations of Asp(813), Asp(815), and Asp(818) to Asn interferes strongly with enzyme activation by Ca(2+) binding and formation of phosphorylated enzyme intermediate by utilization of ATP. Enzyme phosphorylation by Pi in the absence of Ca(2+) undergoes slight or no inhibition by the triple aspartate mutation. Therefore, the triple mutation interferes mainly with the calcium-dependent activation of the ATPase. The effect of the triple mutation can be to a large extent reproduced by single mutation of Asp(813) (but not of Asp(815) or Asp(818)) to Asn. Functional and structural analysis of the experimental data demonstrates that the L67 loop plays an important role in protein folding and function. This role is sustained by linking the cytosolic catalytic domain and the transmembrane Ca(2+) binding domain through a network of hydrogen bonds.