We have investigated the role of eukaryotic initiation factor 2alpha (eIF2alpha) in two estivating organisms previously shown to downregulate protein synthesis during metabolic depression, the land snail Helix aspersa Müller and the desert frog Neobatrachus sutor Main 1957. We have developed a method using a single antibody (which binds specifically to the phosphorylated, conserved phosphorylation region) by which the total levels of eIF2alpha and the ratio of phosphorylated eIF2alpha [eIF2alpha(P)] to total (phosphorylated and unphosphorylated) eIF2alpha can be determined. In H. aspersa, we have shown that the level of eIF2alpha mRNA expression is unchanged between the awake and estivating states. The amount of total eIF2alpha is the same in the estivating and awake states, and eIF2alpha(P) is undetectable and must represent < or =10% of total eIF2alpha in both states. Conversely, in N. sutor during estivation, the level of total eIF2alpha increases approximately 1.6-fold and the ratio of eIF2alpha(P)/eIF2alpha increases from 0.22+/-0.11 to 0.52+/-0.08, implicating eIF2alpha phosphorylation in the downregulation of protein synthesis during estivation in this animal. The differences in the amounts of eIF2alpha and the level of its phosphorylation between these two species also suggest possible differences either in the mechanism by which protein synthesis is downregulated during estivation or in the sensitivity of the initiation of translation to eIF2alpha(P) levels.