The possible causes of the variability of gill metallothionein (MT) levels were studied on 182 specimens of 2- and 3-year old European chub (Squaliuscephalus L.) from the Sava River in Croatia. The most pronounced differences in MT levels were obtained between three sampling campaigns, and especially between periods with presumably different metabolic activities (April/May 2006 vs. September 2005 and 2006). Next to the probable influence of metabolic activity on MT level, the correlation analysis indicated a significant association between MTs and the fish size. Differences between males and females, as well as between mature and non-mature fish, were not observed in the young group of studied chub even in the spring reproductive season. Based on the analysis of the site-specific MT variability, it could be concluded that, under the conditions of low dissolved metal concentrations in the river water (as reported for the Sava River), MTs seem to be more affected by different biotic factors than by metal exposure. Therefore, MTs measured in this study were considered as the constitutive gill MT levels of young European chub. The constitutive MT ranges were defined separately for the season of lower metabolic rate (autumn: 1.31-2.00 mg g(-1)) and the season of higher metabolic activity (spring: 2.15-2.95 mg g(-1)).