The interactions between adsorbed layers of insulin on smooth hydrophilic mica surfaces were measured in situ with a surface force apparatus. Both human insulin and the biologically active monomeric analogue B9Asp + B27Glu were used. In the presence of human insulin, the thickness of the adsorbed layers corresponds to that of a monolayer of hexamers on each mica surface, whereas for the monomeric analogue less than a monolayer of monomers is adsorbed. At large separations an electrical double-layer repulsion dominates the interaction between the protein layers. It is shown that despite the complicated nature of aqueous protein solutions, real insight into the nature of electrostatic forces can be obtained. In particular, in these mixed asymmetric/symmetric electrolytes, the measured values of the decay length depart from the classical Debye screening length based on the ionic strength. A method for extraction of a net effective charge of the proteins in solution is developed, and is compared with other techniques for such determination. The experimental results are discussed in relation to the charge, the association pattern of the protein, the total bulk protein concentration, and the effects of solution properties on both interactions and adsorbed layers. (C) 1994 Academic Press, Inc.