Sealed, inside-out red cell membrane vesicles (IOV) from rats were prepared by a modified method of Steck (1974). The preparation contained 70% of IOV. In the presence of ATP and Mg2+, these vesicles actively took up Ca2+ with a high efficiency and reproducibility. The intraassay coefficient of variance (C.V.) was 3.6% and the interassay C.V. was 13.58%. The active Ca2+ transport reached the maximal level in 10 minutes of incubation, and linearly correlated with the IOV concentration. The Ca2+ uptake rate in Wistar rats was 9.39 +/- 1.28 nmol/mg IOV protein/min. IOV rapidly lost Ca2+ when A23187 was added. The active transport was stimulated by calmodulin, and inhibited by trifluoperazine.