Over a 5-year period, from 1996 to 2001, blood selenium levels were recorded in a set of 2414 healthy blood donors (1781 men and 633 women; 880 smokers and 1534 nonsmokers) living in four selected areas of the Czech Republic. About 100 blood samples per year and region were analyzed using the HGA method. The internal and external quality controls of this method were performed throughout the duration of the study. In general, blood selenium concentrations (81.9 and 106.7 microg/l for median and 90th percentile, respectively) did not reach optimum values; in approximately 10% of the population sample, values lower than 60 microg/l were detected. Nonetheless, the values obtained increased significantly, with median concentrations of 73.2 microg/l in 1996 and 91.5 microg/l in 2001. The percentage of subjects with a whole blood selenium level of less than 60 microg/l also decreased from nearly 20% in 1996 to 0.2% in 2001. No substantial regional or gender-related differences were observed, but significantly lower blood selenium levels were found in smokers as opposed to nonsmokers. Although mild selenium deficiency continues to be observed, the data presented do not indicate extremely low selenium levels in the population sample.