Phospholamban (PLB) is a 25 kDa cardiac regulator protein of the sarcoplasmic reticulum calcium ATPase pump (SERCA2a) which enables cellular relaxation by sequestering calcium within the sarcoplasmic reticulum (SR) whose function is altered during β-AR activation (MacLennan and Kranias, 2003). In its normal dephosphorylated state, PLB inhibits the activity of the SR calcium ATPase re-uptake pump however, upon phosphorylation by protein kinase A or calcium/CaM kinase II, the inhibition exerted by PLB is relieved and cardiac relaxation is enhanced (MacLennan and Kranias, 2003). Studies have reported on Western blotting quantification of both the 25kDa pentameric (Bokník et al. 1999) and also the 6kDa monomeric forms of this protein, where the latter was obtained by heating up the cardiac tissue homogenates to 95°C (Wegener and Jones, 1984). However, one of the suppliers of PLB antibodies for this purpose specifically states that the cardiac samples should not be boiled when probing for PLB (Most et al. 2003). Therefore the objective of this study was to investigate whether there were any significant differences in PLB protein recovery from cardiac tissue homogenates upon boiling or not boiling the cardiac preparations obtained from ventricular tissues of male Wistar-Kyoto (WKY) rats. Western blotting was carried out on left and right ventricular tissue homogenates from WKY rats where one group of homogenates (n = 5) was subjected to 95°C and the other group (n = 5 ) subjected to 37°C for 5 mins before being loaded onto polyacrylamide gel (15‰) and transferred onto a PVDF membrane. The membrane was specifically labelled with the antibody for PLB (1 : 5000) using a polyclonal IgG anti-mouse (1 : 5000) secondary antibody and the recovered protein was quantified using a Bio-Rad G-800 densitometer. Quantification of PLB protein abundances normalised with GAPDH showed no significant differences between the samples heated to 95°C and those heated to 37°C. Furthermore, abundances of monomeric and pentameric forms of PLB when added together for both left and right ventricular samples did not show significant differences. Therefore, this study has shown that PLB can be quantified by assaying both the monomeric and pentameric forms of this critical cardiac regulatory protein and there is no significant differences in PLB recovery from cardiac homogenates undergoing boiling or not as a pre-treatment.