The binding of 6 alpha-methylprednisolone and 6 alpha-methylprednisone to proteins of rabbit and human plasma was studied in vitro by equilibrium dialysis. Steroid binding was determined using radiolabeled compounds and HPLC analysis methods. Both methods produced equivalent results. Plasma protein binding of 6 alpha-methylprednisolone and 6 alpha-methylprednisone averaged 75-82% and was independent of steroid concentration, suggestive of low affinity, nonspecific protein binding. A positive linear correlation of the log octanol-water partition coefficient with the nonspecific binding affinities of a homologous series of steroids, including 6 alpha-methylprednisolone and 6 alpha-methylprednisone, was demonstrated. This correlation suggests that hydrophobic binding is a major determinant of nonspecific steroid-protein interactions.