Four different isoforms of the membrane sodium-hydrogen exchanger (NHE-1 to 4) have been identified and cloned. NHE-1 mRNA is ubiquitously expressed, whereas isoforms NHE-1 to 3 exhibit a more limited distribution amongepithelial cells. Although physiologic experiments demonstrate the existence of a sodium-hydrogen exchange mechanism in platelets, the structural identity remains to be determined. We sought to resolve whether platelets express any of the genes for NHE isoforms. Total RNA was isolated by a modified guanidinium isothiocyanate method from mature platelets, anucleate cells not capable of RNA synthesis de novo. Oligonucleotide primers for PCR amplification of regions contained within the cytoplasmic regulatory loop of NHE isoforms were employed in RT-PCR amplification of cDNAs synthesized from platelet RNA. Fragments of 862 and a 884 bp were detected for NHE-1 and NHE-3 respectively. Extending our observations in platelets, we further explored the hypothesis that NHE-3 might also be expressed in other cells of hematopoietic cell lineage. Similar results were obtained in human leukocytes, using the NHE primers. The PCR products were the same size as those synthesized from rat small intestine and kidney RNA's, tissues previously demonstrated to express NHE-3. This report is the first demonstration of the presence of NHE-3, an“epithelial specific” isoform, in non-epithelial cells. Using sensitivity to amiloride as a physiologic parameter to differentiate between NHE-1 and other isoforms in a platelet aggregation assay, we are currently testing the functional importance of NHE-3 in platelets by determining the extent to which sodium loading of platelets enhances platelet activation. We conclude that platelets express two different isoforms of NHE: an amiloride sensitive form NHE-1 and amiloride resistant form which is NHE-3, based upon our molecular analysis. The relative functions of each of these NHE isoforms in platelets remain to be elucidated. (This study was supported by NIH GM46588 to MJE).