Detection and assessment of the impact of pollution on biological resources imply increasing research on early-warning markers such as metallothioneins in metal exposure. Metallothioneins are cytosolic, low molecular weight proteins, involved principally in essential metal homeostasis and non-essential metal detoxication. Metallothionein synthesis could be influenced by abiotic (season) or biotic (reproduction process) factors directly or indirectly by its effect on metal bioaccumulation (i.e., sex, weight). In a view of using metallothioneins as metal-exposure biomarkers in Gammarus pulex, this study attempts to define the effect of several factors (sex, weight/size and season) on the level of this protein. Metallothionein levels recorded in individuals over a large range of weights indicate a negative correlation between them. Inversely in our conditions, no difference was observed between male and female organisms. During field study, metallothionein level changes were observed with the highest levels in autumn and winter periods. The highest metallothionein levels were observed after the reproduction period, perhaps linked with the metabolic needs of biologically available essential metal such as zinc.