Platelet-endothelial cell adhesion molecule-1 (PECAM-1) is an integral component of endothelial cells and has been implicated in the transendothelial migration (TEM) of circulating leukocytes mediated by its 1st and 2nd extracellular immunoglobulin (Ig)-like domains and regulation of intracellular Ca2+ homeostasis with its 6th domain. Up-to-date, little is known about the role of the 5th extracellular (Ig)-like domain. We have discovered a novel human PECAM-1 transcript missing the entire 7th exon, which encodes the 5th extracellular (Ig)-like domain of PECAM-1. A synthetic peptide with sequence homology to the 5th domain of PECAM-1 (JHS-7 peptide) and a corresponding polyclonal antibody (JHS-7 Ab) were prepared and their potential role in transendothelial migration and Ca2+ influx was measured. The JHS-7 peptide and the antibody exerted a dose dependent decrease (50-80%) in the transendothelial migration of freshly isolated human monocytes and a promonocytic cell line (U-937) in resting HUVECs and HUVECs activated with tumor necrosis factor-alpha. This was accompanied by an increase in Ca2+ influx and decrease in refilling of the intracellular Ca2+ stores in HUVECs. In summary, we have identified a novel PECAM-1 transcript (Deltaexon 7) and shown that the 5th (Ig)-like domain of PECAM-1 plays a role in monocyte TEM and Ca2+ homeostasis. (C) 2004 Elsevier Inc. All rights reserved.