Electron paramagnetic resonance (EPR) spectroscopy has been used to characterize the binding of spin-labeled fatty acids (SLFA) to two isolipases (lipases A and B) from Candida rugosa. The spectra of the bound species obtained with 5-doxyl and 16-doxyl stearic acids (5-DSA and 16-DSA) at a low molar ratio of fatty acid to lipase (0.15/1) have been analyzed using slow motion EPR simulations. Analysis of the EPR parameters extracted from the simulations (rotational diffusion coefficients, R-parallel to, R-perpendicular to, and R) supports the existence of a tunnel in the enzyme which imposes constraints on the motion of the SLFA. These constraints depend on both the nature of the lipase (there is higher mobility of the label in lipase A than in Lipase B) and the position of the label (the mobility at C-16 of the SLFA is higher than at C-5 for lipase A). With respect to both the C-5 and C-16 positions of the bound SLFA, the polarities of both isolipases (measured from the isotropic hyperfine splitting, A(o), in the low temperature spectra) are essentially the same. This result implies nearly identical polarities at the substrate binding region of these enzymes. However, the polarity at the C-5 position is greater than that at C-16. The polarity data lead one to the conclusion that differences in the activities of C. rugosa isolipases are not a consequence of differences in the polarity of the protein binding region. The lower specific activity of lipase A relative to that of lipase B for the hydrolysis of triacylglycerols has been interpreted in terms of the mobility and binding data for the two SLFA＇s. Binding studies (Scatchard analysis) involving different molar ratios of fatty acid to lipase (0.1/1-5/1) for 5-DSA and 16-DSA indicate the presence of four interdependent binding sites for these substrates in the isolipases from C. rugosa. For both isolipases, the association constants are of the order of 10(4), a value which is characteristic of specific ligand-protein interactions. The binding constant for isolipase A is higher than that for B. In terms of the cooperative character of the binding, the interactions of these fatty acid analogues with C. rugosa isolipases are consistent with a conformational rearrangement of the hydrophobic pocket of the protein.