The lipid raft concept proposes that membrane environments enriched in cholesterol and sphingolipids cluster certain proteins and form platforms to integrate cell signalling. In cardiac muscle, caveolin-enriched rafts concentrate signalling molecules and ion transporters, and therefore play a vital role in adrenergic regulation of cardiac excitation-contraction coupling. The aim of this project was to define the proteome of cardiac lipid rafts, and to investigate dynamic changes in the protein constituents of rafts following activation of α1, β1 and β2 adrenoceptors (AR). Calcium-tolerant ventricular myocytes (VM) were isolated from the hearts of adult male Wistar rats by retrograde perfusion of collagenase. Myocytes were left to recover for 2 hours at 35oC before experiments. VM were treated with methyl-β-cyclodextrin (MβCD) to deplete cholesterol and disrupt lipid rafts. Rafts were prepared from MβCD-treated and control cell lysates using a standard discontinuous sucrose gradient. Buoyant rafts were harvested, pelleted, resolubilised, alkylated, digested and labeled with iTRAQ reagents, and proteins identified by LC-MS/MS on a LTQ Orbitrap Velos Pro. Proteins were defined as lipid raft resident if they were consistently depleted from the raft fraction following MβCD treatment, and as contaminants (principally mitochondrial) if they were not. We typically identified 600-850 raft proteins per experiment, of which 254 were defined as high-confidence lipid raft residents. Functional annotation clustering indicates that cardiac lipid rafts are enriched in integrin signalling, guanine nucleotide binding, ion transport, and insulin signalling clusters. The presence of selected proteins of each group in the lipid rafts was validated by immunoblotting. In order to define changes in cardiac lipid rafts during adrenergic signalling, field-stimulated VM were treated with α- (10µM phenylephrine, 10µM atenolol), β1- (100nM isoprenaline, 100nM ICI118,551) and β2- (10µM zinterol, 300nM CGP20712A) AR agonists for 10 min prior to lysis and preparation of lipid rafts. Following iTraq analysis, raft resident proteins showing a change in abundance in a minimum 3 out of 4 experiments were selected. β1 AR activation is associated with a decrease in abundance of PKA RIIα and an increase in abundance of cavin isoforms in cardiac lipid rafts. β2 AR stimulation increases the abundance of G protein and L-type Ca channel subunits in cardiac rafts. In conclusion, we have identified 254 lipid raft proteins in adult rat cardiac myocytes and shown for the first time that these domains are re-organised on an acute timescale in response to β-AR stimulation. Dynamic changes in certain raft components may be necessary to ensure a rapid and high fidelity response to adrenergic stimulation in cardiac muscle.