Saliva collected from volunteers of both sexes at various times throughout the day was examined for the presence of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3. The method used was a specific high-performance liquid chromatographic procedure with a minimum detectable limit for 25-hydroxyvitamin D of 0.05 microgram/1, (130 pmol/1 for 25-hydroxyvitamin D3) using 20 ml of saliva. No 25-hydroxyvitamin D could be detected in any of the samples. Examination of larger quantities of saliva (100 ml) by a combination of high-performance liquid chromatography and gas chromatography-mass spectrometry showed the presence of a peak possibly derived from 25-hydroxyvitamin D3, at a concentration not in excess of 65 pmol/l. Using known binding characteristics of 25-hydroxyvitamin D and plasma vitamin D binding protein, and comparing them with similar characteristics for cortisol and plasma cortisol binding globulin and the known salivary concentration of cortisol, simple calculations indicated that a likely concentration of 25-hydroxyvitamin D in saliva would be around 8 pmol/l. Previously reported values ranging from 130-4160 pmol/l (50-1600 micrograms/l) using a simple competitive protein binding assay could not be confirmed.