Introduction: Embryonic rat aortic smooth muscle cells, A7r5, have been used extensively as an in vitro vascular smooth muscle cellmodel. They are usually provided at 11th passage by supplier and generally used before 25th passage. However, the exact passage number (P#) used is not reported in general. Methods: In this study, A7r5 cells (P# < 18 and P# > 23) were used in quantitative gene (5-HT2A and SERCA2b) expression as well as in functional analyses including measurements of real-time changes in cell proliferation and in 5-HT-and CPA-induced intracellular Ca2+ levels. Results: CPA-induced SR Ca2+ release and store-operated Ca2+ entry significantly increased (p < .05) while cell proliferation decreased in P# > 23 cells (p < .01). Furthermore, 5-HT-induced Ca2+ elevations and 5-HT2A mRNA levels did not change whereas SERCA2b mRNA levels and CPA-induced [ Ca2+](i) levels were significantly elevated in P# > 23 cells (p < .05, p < .01, respectively). Discussion: Changes in SERCA2b expression and SOCE may contribute to suppression of cell proliferation during A7r5 subculturing. Therefore, passage numbers and subculturing procedure should be reported and taken into account during expressional and functional analyses for an accurate comparison of published data. (C) 2014 Elsevier Inc. All rights reserved.