Several pollutants have the potential to disrupt the endocrine system in aquatic organisms, and synthesis of vitellogenin (VTG) in male fish is a well-recognized effect of estrogenic xenobiotics. In this respect both the presence of the protein in plasma and the analysis of VTG gene induction may represent valuable biomarkers. The present article describes primers specifically designed for a RT-PCR assay of VTG mRNA in various Mediterranean fish species. All the species analyzed have great potential as bioindicators in the Mediterranean: the red mullet (Mullus barbatus) and the striped mullet (Mugil cephalus) are commonly found in coastal and estuarine waters, the black goby (Gobius niger) is an important species in harbors, the European eel (Anguilla anguilla) is more typical of brackish environments and lagoon ecosystems, and the tuna fish (Thunnus thynnus) has commercial value and, being a top predator in marine food webs, is particularly exposed to bioaccumulated halogenated hydrocarbons with possible estrogenic activity. The analysis of VTG mRNA has been standardized in feral fish, and basal expression of VTG was demonstrated in female specimens of the species analyzed. Only sexually immature specimens were analyzed for A. anguilla, and exposure to 17β-estradiol clearly induced the synthesis of VTG mRNA, confirming their responsiveness to estrogenic exposure and the specificity of the designed primers. VTG mRNA was detected in adult males of T. thynnus (>100kg), supporting estrogenic exposure of older specimens. In this species two different VTGs were identified, and the sequences obtained in the various species were compared with available sequences.