Background: Balkan endemic nephropathy (BEN) has not been described in children, however some previous studies of children from families with BEN have revealed abnormalities of the urinary tract including an increased urinary protein excretion. Methods: In the present study, urinary excretion of total protein was studied in 703 healthy children, aged 9–13, from endemic and non-endemic settlements around the South Morava River. Since BEN is an environmentally induced disease, with possible seasonal variation of toxin(s), children were studied three times a year: in spring, autumn and winter, during a 3-year period. After a water load of 15 ml/kg body weight, a 3-hour urine sample was collected, from 7 to 10 a.m. Results: Protein excretion in urine was highest in children from families with BEN compared with the excretion in children from the city, non-endemic villages, and those from non-endemic families living in the same settlements. This was the case during all three periods investigated in the second year of the study. In the autumn term of all three years of the study, protein excretion was significantly higher in children from families with BEN than in children from the city and from non- endemic families living in the same settlements. If the upper limit of protein excretion is set at 34 mg/mmol creatinine, then increased protein excretion in autumn was found in the first year of study in 9/229 children from endemic settlements and in only 4/474 children from non-endemic areas (p = 0.004); in the second year in 5/229 and 3/474 children (p = 0.069), and in the third year in 10/229 and 4/474 children (p = 0.002), respectively. Conclusions: We have presented evidence that children from families with BEN and endemic villages consistently excreted significantly more protein in the autumn, and in three seasons (spring, autumn, winter) in 1 year of our study. These results are consistent with seasonal variations in exposure to an environmental toxin(s).