The binding of the 1-anilinonaphthalene-8-sulfonate(ANS) to bovine serum albumin was studied by fluorescence spectroscopy. The effect of pH, ionic strength, and protein concentration on the binding of ANS to protein were compared. The binding between ANS and protein was dependent on pH and ionic strength. It seems that both hydrophobic binding and some electrostatic forces are involved in the binding of ANS to protein. The binding constants for ANS increased with increasing protein concentration. This suggests the possibility of a sharing of one ANS molecule by more than one protein molecule at relatively high protein concentration.