Many defined cell culture media were formulated over 3() years ago and may be deficient in certain micronutrients whose essentiality has only subsequently been recognised. The objective of this study was to evaluate whether alpha-minimal essential medium (MEM) supplemented with 10% foetal bovine serum contained sufficient selenium for optimal activity of the selenium containing enzymes cytosolic glutathione peroxidase (cGPx) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) in cultured Chinese hamster ovary (CHO) cells. Additionally, the effect of zinc deficiency and metallothionein (MT) overexpression on cGPx and PHGPx activity was studied. The addition of 100 nM of selenous acid to the culture medium increased cGPx expression by 10-fold and PHGPx by about 2-fold in both wild-type CHO-K1 cells and CHO-K1 cells overexpressing mouse MT-1. Zinc deficiency had no significant effect on enzyme activity, but cells overexpressing mouse MT-1 had higher levels of cGPx activity. Zinc deficiency decreased cell survival but overexpression of MT-1 was partially protective, probably because its presence in quantity favoured the uptake, sequestration and cellular retention of any remaining zinc. This study demonstrates that selenium in complete alpha-MEM is insufficient for optimal cGPx and PHGPx activity and may compromise the cellular response to oxidative stress.