Most of the total circulating 1,25-dihydroxyvitamin D (1,25(OH)2D) is bound to plasma proteins, mainly vitamin D-binding protein (DBP) and albumin. Only a small fraction in plasma exists in the free form. It is widely assumed that the non-protein-bound free hormone reflects the biologically active fraction. We describe a dialysis method for the determination of plasma free 1,25(OH)2D which is relatively easy to perform. In this symmetric or "rate" dialysis method, identical samples are placed at both sides of a membrane. At one side, tritiated 1,25(OH)2D is added and the rate of transfer of this tritiated 1,25(OH)2D through a dialysis membrane is directly related to the free fraction of plasma 1,25(OH)2D. This method is much less susceptible toward tracer impurities than indirect equilibrium dialysis and centrifugal ultrafiltration. Moreover, it requires much less tracer. The intraassay coefficient of variation for the determination of the free fraction is 1.0%; the interassay variation is 7.7%. Comparison of the free fraction of 23 samples assessed with both centrifugal ultrafiltration and symmetric dialysis showed much higher values using the former method. No significant correlation between the two methods was found. The free fraction of 1,25(OH)2D in normal subjects as assessed with symmetric dialysis ranges from 0.049 to 0.103%.