Several groups have addressed the issue of the influence of GC on expression levels in mammalian genes. In general.. GC-rich genes appeared to be more expressed than GC-poor ones. Recently, expression levels of GC(3)-rich and GC(3)-poor versions of genes (GC(3) is the third codon position GC), inserted in vector plasmids, were compared in order to eliminate differences associated with their genomic context. Transfection experiments showed that GC(3)-rich genes were expressed more efficiently than their GC(3)-poor counterparts, indicating that GC3 dramatically and intrinsically boosts expression efficiency. Here we show that, while the protocols used eliminated the original genomic context, they replaced it with the plasmid contexts whose compositional properties affected the results. (C) 2007 Elsevier Inc. All rights reserved.