The forces between proteoheparan sulfate layers adsorbed at hydrophobic surfaces were investigated by direct surface force measurements. In 0.2 mg/mL proteoheparan sulfate and 0.1 mM NaCl, the forces were monotonically repulsive, with one distant regime having a decay length of about 180 angstrom, and one steeply repulsive proximal part at distances smaller than about 100 angstrom. Only small effects were observed on dilution with 0.1 mM NaCl, indicating "irreversible" adsorption. The decay length of the distant force component was 280 angstrom after dilution, whereas the proximal repulsive component remained unchanged. Furthermore, after dilution, a weak adhesion (FIR = 200 muN/m) was observed. On addition of CaCl2, the decay length of the distant repulsion decreased from 280 angstrom in 0.1 mM NaCl to 86 and 77 angstrom in 1.25 and 2.5 mM CaCl2, respectively. The decay lengths in CaCl2 solutions are significantly larger than the expected Debye lengths, demonstrating the predominance of steric forces. Furthermore, on addition of CaCl2 the magnitude of the adhesion increased from 200 to 1000 muN/m, and the proximal repulsion was observed at slightly smaller distances. It was found that both steric and electrostatic forces contribute to the interactions between proteoheparan sulfate layers.