We have examined the fluorescence properties and acrylamide quenching of calcium-loaded (holo) and calcium-depleted (apo) a-lactalbumin ((U-LA) as a function of guanidine hydrochloride (GDN/HCl) concentration. The spectral changes accompanying increasing GDN/HCl are consistent with protein unfolding and a release of internal fluorescence quenching, which occurs among the three tryptophan residues located in the region of the so-called "tertiary fold." Values for the intrinsic fluorescence emission, the wavelength maximum of the emission, the Stern/Volmer dynamic quench constant, and the static quench constant are consistent with a significant stabilization effect by calcium against protein unfolding. The dynamic quench constant of apo-alpha-LA increases fourfold to its maximum, in the transition from the native state to protein in 1.5 M GDN/HCl, The dynamic quench constant for holo-alpha-LA remains unchanged until exposed to 2.5 M GDN/HCl, but increases by threefold with addition denaturant to 4 M GDN/HCI. The static quench constant of the ape-protein in the native solvent, approximately 0.2 M-1, declines to zero in 1 M denaturant, where the molten globule folding intermediate is most populated. A more protracted denaturant-dependent decline in the static quench constant occurs for the holoprotein. Sharp increase in the static quenching occurs for ape-a-LA and holo-cu-LA above 1.5 M GDN/HCI and 3.5 M GDN/HCl, respectively. The results for apo-alpha-LA in dilute GDN/HCl suggest that acrylamide can penetrate the protein molecule las judged by the collision quenching) but is unable to form a stable complex within the quenching domain for the tryptophans (as judged by the absence of the static quench constant). It seems reasonable to suggest that the protein folding intermediate which occurs in dilute denaturant represents a structure in which the tryptophans are, on average, more accessible to collisional quenching but sufficiently compact to prevent formation of a stable, dark equilibrium complex with acrylamide.