The effects of temperature and protein concentration on the binding of thiopental to bovine and rat serum albumin and to rat plasma were examined using flow and equilibrium dialysis techniques. The effects of temperature and protein concentration were peculiar to each species. The binding of thiopental to rat serum albumin or rat plasma was sensitive to temperatures between 4 and 37 degrees, whereas the binding to bovine serum albumin was sensitive to a protein concentration difference of 0.1-4.44%. Therefore, the effective protein fraction is affected by various sets of binding parameters of albumins determined under various temperatures and protein concentrations. Thus, a semi-predictive method for plasma or tissue binding may be unsuccessful unless proper binding parameters are used.