The concentration of testosterone in whole saliva is significantly increased (by 9%) after toothbrushing. In ultrafiltrates of saliva collected at the same time as the whole saliva, testosterone concentrations after toothbrushing were unchanged. In 88% of the 162 whole-saliva specimens, but not in the ultrafiltrates, we also measured higher hemoglobin concentrations after toothbrushing. We conclude that the increase of testosterone in whole saliva after toothbrushing can be attributed to a protein-bound fraction. For analytes that are bound to serum proteins, salivary measurements can give spurious results. This problem can be avoided by using as a diagnostic medium an ultrafiltrate of saliva collected directly in the mouth.