We determined the serum concentrations of 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D [1,25-(OH)2D], 24,25-(OH)2D, and 25,26-(OH)2D every third month in a 2-yr longitudinal study of 20 normal pubertal boys. Seasonal fluctuations and highly significant correlations with the hours of sunshine were found for 25-hydroxyvitamin D, 24,25-(OH)2D and 25,26-(OH)2D, whereas the serum concentrations of 1,25-(OH)2D were not related to sunshine duration. The boys were grouped according to the time at which the maximal increase in serum testosterone occurred, and the serum vitamin D metabolite levels were examined in relation to this point. No significant changes in the serum levels of the vitamin D metabolites were found. When compiling the data according to clinical evaluation of puberty estimated by pubic hair stages (PH1 to PH4), no significant changes in the serum levels of vitamin D metabolites were found. The present data indicate that the marked increase in serum testosterone in male puberty has no significant influence on circulating vitamin D metabolite levels.