Mussels are widely used as bioindicator organisms for monitoring chemical pollutants including trace metals. These elements are natural constituents in the marine environment and their basal concentrations in the organisms can be influenced by several environmental and biological factors. The aim of this work was to extend our knowledge on the natural variability of trace metals in mussels tissues, focusing on seasonal and inter-annual fluctuations in a coastal reference site of the Adriatic coast (Portonovo); a total of 39 samplings were performed during 5 years, providing an extended data-set for tissue levels of As, Ba, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, V and Zn. Concentrations of trace metals in mussels tissues revealed marked seasonal fluctuations with significant differences between various sampling years. Such fluctuations appeared mostly related to phytoplanctonic blooms and especially to reproductive cycle which exhibited a certain inter-annual shift of the gametogenesis period. Lower concentrations were measured in summer months for the majority of elements while a different seasonal cycle was observed for arsenic, not correlated with gonadic development, neither with other elements. Chemical speciation of arsenic was characterized to distinguish compounds of natural origin from those potentially reflecting an anthropogenic impact. Arsenobetaine and arsenocholine were always the predominant forms (up to 85% of total arsenic), while a significant increase of dimethylarsine and trimethylarsine oxide in spring (24% of total arsenic) might reflect the effect of phytoplanctonic bloom on both geochemistry and trophic transfer of this element. A significant inter-annual variability was observed for both the seasonal cycle and the range of values measured for all the elements, with particularly marked differences for arsenic, ranging from less than 10 to more than 40 microg/g in summer periods of different years. Data obtained on reference mussels were used to assess the impact of 41 off-shore platforms distributed along 5 nautical districts in the Northern and Central Adriatic Sea. Organisms sampled on these structures from 2001 to 2005 exhibited a certain enrichment of cadmium and zinc, probably associated to the use of anodic electrodes. Marked annual and geographical variations were measured for concentrations of arsenic in mussels of different platforms. However, the comparison with results of Portonovo, allowed to exclude the anthropogenic impact of exploitation activities and revealed a natural regional gradient of arsenic levels in mussels tissues associated to the changing influence of the Po river runoff on seawater salinity. In particular the higher concentrations measured in organisms sampled from platforms in the Central compared to Northern Adriatic confirmed a significant relationship between salinity and arsenic bioaccumulation, consistent with a role of arsenobetaine as an acquired osmolyte for mussels. The overall results confirmed the importance of natural variability when assessing the potential impact of anthropogenic activities.