ATP utilization by P-type cation transport ATPases includes a phosphorylated intermediate which is formed by transfer of the ATP terminal phosphate onto an aspartyl residue at the catalytic site. The phosphorylation site and cation binding site of the ATPase molecule are separated by a fairly long distance of about 50 A. The coupling mechanism of these two functional sites is not yet fully understood and is currently under active investigation. Within the family of cation transport ATPases, the Ca2+-ATPase of sarcoplasmic reticulum (SR) provides an advantageous experimental system due to its abundance in the native membrane, and the availability of cDNA for expression of functional protein.;The sarcoplasmic reticulum Ca2+-ATPase segment extending from the phosphorylation site (Asp351) to the preceding transmembrane helix M4 (which is involved in Ca2+ binding in conjunction with transmembrane helices M5, M6 and M8), shares a marked sequence homology with the corresponding segments of other cation ATPases. We generated twenty six point mutations in this segment and expressed those mutant enzymes in COS-1 cells. We found that non-conservative mutations of residues which are homologous in various cation ATPases result in strong inhibition of catalytic and transport functions. Mutations of non-homologous residues to match the corresponding residues of other cation ATPases are not inhibitory, and in some cases produce higher activity. The inhibitory mutations specifically affect the phosphorylated intermediate turnover, which is associated with the vectorial translocation of bound Ca2+. The same mutations do not affect the kinetics of ATPase activation by Ca2+, which is required for enzyme phosphorylation by ATP. This indicates that activation of the phosphoryl transfer reaction by Ca2+ binding, and vectorial displacement of bound Ca2+ by enzyme phosphorylation, do not occur simply as the forward and reverse directions of the same process, but are linked to distinct structural features of the enzyme. The peptide segment extending from the phosphorylation site in the enzyme extramembranous headpiece, through the M4 helix in the membrane bound region, sustains a prominent role in transmission of the phosphorylation signal for displacement of bound Ca2+. A critical structural role of this segment is also demonstrated by the interference of specific mutations with membrane assembly of the expressed protein.