The 31-amino acid proteolipid, sarcolipin (SLN), is associated with the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase (SERCA1). Constructs of human and rabbit SLN and of rabbit SLN with the FLAG epitope at its N terminus (NF-SLN) or its C terminus (SLN-FC) were coexpressed with SERCA1 in HEK-293 T-cells. Immunohistochemistry was used to demonstrate colocalization of NF-SLN and SERCA1 in the endoplasmic reticulum membrane and to demonstrate the cytosolic orientation of the N terminus of SLN. Coexpression of native rabbit SLN or NF-SLN with SERCA1 decreased the apparent affinity of SERCA1 for Ca2+ but stimulated maximal Ca2+ uptake rates (Vmax). The N terminus of SLN is not well conserved among species, and the addition of an N-terminal FLAG epitope did not alter SLN function. Anti-FLAG antibody reversed both the inhibition of Ca2+ uptake by NF-SLN at low Ca2+ concentrations and the stimulatory effect of NF-SLN on Vmax. Addition of the FLAG epitope to the highly conserved C terminus decreased the apparent affinity of SERCA1 for Ca2+ relative to native SLN and decreased Vmax significantly. Mutations in the C-terminal domain showed that this sequence is critical for SLN function. Mutational analysis of the transmembrane helix, together with the additive regulatory effects of coexpression of both SLN and phospholamban (PLN) with SERCA1, provided evidence for different mechanisms of interaction of SLN and PLN with SERCA molecules. Ca2+ uptake rates in sarcoplasmic reticulum vesicles, isolated from rabbit fast-twitch muscle (tibialis anterior) subjected to chronic low frequency stimulation, were reduced by approximately 40% in 3- and 4-day stimulated muscle, with a marginal increase in apparent affinity of SERCA1 for Ca2+. SERCA1 mRNA and protein levels were unaltered after stimulation. In contrast, SLN mRNA was decreased by 15%, and SLN protein was reduced by 40%. Reduced SLN expression could explain the decrease in SERCA1 activity observed in these muscles and might represent an early functional adaptation to chronic low frequency stimulation.