A mutant of the plasma membrane Ca2+ pump (PMCA) called (nCI)hPMCA4b(ct120), in which the C-terminal regulatory segment including the calmodulin-binding autoinhibitory domains C and I had been relocated near the N-terminus, has been expressed in COS-1 cells. The measurements of Ca2+ transport in microsomal preparations showed that the rearranged enzyme was functional. The activity of the (nCI)hPMCA4b(ct120) mutant was compared with those of the wild-type hPMCA4b and the fully active calmodulin-insensitive mutant hPMCA4b(ct120). In the absence of calmodulin the activity of (nCI)hPMCA4b(ct120) was higher than that of hPMCA4b but only 45% of that of hPMCA4b(ct120). Mutant (nCI)hPMCA4b(ct120) exhibited an apparent affinity for Ca2+ similar to that of hPMCA4b, typical of the inhibited state of the enzyme. Calmodulin at concentrations that fully activated hPMCA4b increased the activity of (nCI)hPMCA4b(ct120) to 68% of that of hPMCA4b(ct120). The lower maximal activity of (nCI)hPMCA4b(ct120) was not due to a lower affinity for calmodulin because the concentration of calmodulin required for half-maximal activation of (nCI)hPMCA4b(ct120) was equal to that of the wild-type hPMCA4b. These results indicate that (1) the disturbance of the N-terminal region of the PMCA by the insertion of the C-terminal segment decreased the ability of the pump to transport Ca2+, and (2) the calmodulin-binding autoinhibitory domain was still able to access its acceptor site from the N-terminal end of the molecule. However, although the calmodulin-binding and inhibitory functions of the C-domain were fully preserved, the I domain at its new position seemed less effective at inhibiting the pump.