Interlaboratory variation of the measurement of 25-hydroxy vitamin D, 24,25-dihydroxy vitamin D and 1,25-dihydroxy vitamin D by six laboratories in the Netherlands was studied. Three different serum samples and two different standard solutions of each metabolite were assayed. Substantial interlaboratory variation was found for the measurement of serum samples. The mean interlaboratory CV's for the 25-hydroxy vitamin D, 24,25-dihydroxy vitamin D and 1,25-dihydroxy vitamin D assays in the three sera were 48%, 38% and 20% respectively. The measurement of standard solutions of all metabolites showed relative little variation (mean CV 8%). The small number of samples allowed no evaluation of intralaboratory variation. The much higher CV's of the measurement of serum samples, when compared to standard solutions, may be attributed to differences in extraction and purification procedures which are probably responsible for the presence of varying amounts of interfering substances during the final quantification of metabolites.