Phospholemman (PLM), the principal quantitative sarcolemmal substrate for protein kinases A and C in the heart, regulates the cardiac sodium pump. Much like phospholamban, which regulates the sodium pump-related calcium ATPase SERCA, PLM is reported to oligomerise. We investigated subpopulations of PLM in adult rat ventricular myocytes based on phosphorylation status. Calcium-tolerant ventricular myocytes (ARVM) were isolated from adult male Wistar rats (200-250g) by retrograde perfusion of collagenase in the Langendorff mode. Myocytes were left to recover for 2 hours at 35oC before experiments. Coimmunoprecipitation identified two pools of PLM: one not associated with the sodium pump phosphorylated at serine 63 (S63), and one, associated with the pump, both phosphorylated at serine 68 and unphosphorylated. Phosphorylation of PLM at S63 following activation of PKC did not abrogate association of PLM with the pump, so its failure to associate with the pump was not due to phosphorylation at this site. All pools of PLM co-localised to cell surface caveolin-enriched microdomains with sodium pump α subunits, despite the lack of caveolin-binding motif in PLM. Mass spectrometry analysis of phosphospecific immunoprecipitation reactions revealed no unique protein interactions for S63- phosphorylated PLM, and crosslinking reagents also failed to identify any partner proteins for this pool. The sodium pump α subunit co-immunoprecipitated with PP2A from ARVM lysates. Since PLM-S63 is the only phosphorylation site in PLM dephosphorylated by PP2A, this likely explains the lack of S63-phosphorylated PLM associated with the pump. In lysates from hearts of heterozygous transgenic animals expressing wild type and unphosphorylatable PLM, S63- phosphorylated PLM co-immunoprecipitated unphosphorylatable PLM, therefore confirming the existence of PLM multimers. Hence we report the existence of a subpopulation of PLM that interacts only with other PLM molecules and not with the Na pump, with a unique phosphorylation status driven by differential proximity of protein phosphatases. Like phospholamban regulation of SERCA, PLM exists as a sodium pump inhibiting monomer and an un-associated oligomer. The distribution of different PLM phosphorylation states to different pools may be explained by their differential proximity to protein phosphatases, rather than a direct effect of phosphorylation on PLM association with the pump.