CD4 is a transmembrane glycoprotein expressed on T-lymphocytes. It is a receptor for class II major histocompatibility complex (MHC) molecules and for the HIV envelope glycoprotein gp120. The extracellular portion of CD4 (sCD4) is a rod-shaped molecule consisting of four domains designated DI through D4. Denaturant-induced unfolding of sCD4 and of isolated CD4 domains, D1D2 and D3D4, was measured using both ultraviolet absorbance and fluorescence difference spectroscopy. Though both absorbance and fluorescence changes arise from changes in the solvent exposure of the intrinsic tryptophan chromophores, the unfolding curves obtained with the two techniques looked very different for sCD4 and D3D4. This dissimilarity is indicative of a greater than two-state unfolding mechanism. The global three-state fit for sCD4, which simultaneously fit both absorbance and emission data to a model with one thermodynamically stable unfolding intermediate, was significantly better than the best two-state fit, suggesting that there are two unfolding regions. Unfolding of isolated D3D4 also fit a three-state model while unfolding of isolated D1D2 fit satisfactorily to a two-state model. The unfolding of these two isolated fragments could not be summed to yield the unfolding profile of sCD4, implying that an interaction between D2 and D3 is lost by splitting sCD4 between these domains. The unfolding data of isolated D1D2 and D3D4 were used with the solvent-accessible surface area of tryptophans calculated from atomic crystal structure coordinates of human D1D2 and rat D3D4 to assign the unfolding steps. The data are consistent with a model for sCD4 unfolding wherein the one stable intermediate appears to contain only the D4 domain unfolded. The remaining three domains apparently unfold as a unit. Furthermore, interactions between domains D1, D2, and D3 appear to stabilize D4, suggesting that stabilizing interactions exist between D3 and D4 even though the unfolding of the D3D4 fragment is best fit by a three-state model. This report is the first to describe a thermodynamic basis for a wide range of biological properties implicated for CD4. © 1995, American Chemical Society. All rights reserved.