Owing to their therapeutic relevance, considerable efforts are devoted to the structural characterisation of membrane proteins. Such studies are limited by the availability of high quality protein due to the difficulty of overexpression in recombinant mammalian systems. We sought to systematically optimise multiple aspects in the process of transiently transfecting HEK293 cells, to allow the rapid expression of membrane proteins, without the lengthy process of stable clone formation. We assessed the impact of medium formulation, cell line, and harvest time on the expression of GABA(A) receptors, as determined by [H-3]muscimol binding in cell membranes. Furthermore, transfection with the use of calcium phosphate/polyethyleneimine multishell nanoparticles was optimised, and a dual vector system utilising viral enhancing elements was designed and implemented. These efforts resulted in a 40-fold improvement in GABA(A) alpha(1)beta(3) receptor expression, providing final yields of 22 fmol/cm(2). The findings from this work provide a guide to the optimisation of transient expression of proteins in mammalian cells and should assist in the structural characterisation of membrane proteins.