Revised Manuscript Received February 22, 1988 abstract: Previous folding studies have shown that equilibrium denaturation of bovine growth hormone (bGH) is a multistate process with stable intermediates. The native and unfolded species are monomeric, but intermediates are both monomeric and associated. In this study, the relative insolubility of an associated intermediate is used to distinguish its presence in equilibrium denaturation and during kinetic refolding. To study therole of the associated intermediatein the refolding pathway, a two-step procedure for its detection was developed. The first step of this procedure is used to populate the associated intermediate, and the second step involves dilution to solvent conditions in which only the associated intermediate precipitates. The amount of precipitate is quantitated either directly by formation of turbidity or indirectly by quantitation of the remaining soluble protein. The results show that an associated species is transiently populated during folding, it is incorrectly folded, and it occurs due to specific interactions of monomeric folding intermediates at moderate to high protein concentrations. This association of intermediates is a competing reaction that decreases the folding rate. The location of this competing reaction in the refolding pathway occurs after the formation of an early framework-type intermediate that contains considerable secondarystructure but prior to the rate-limiting formation of the native tertiary structure. When refolding occurs in solutions that solubilize theassociated intermediate, then native protein is obtained quantitatively. However, if refolding occurs in solutions that do not solubilize the associated intermediate, then most of the product results in an insoluble protein aggregate. The formationof precipitate that occurs upon refolding is inhibited by addition of fragments 96-133 or 109-133 that are derived frombGH. It is suggested that these fragments prevent precipitation by binding to the framework-type intermediate in a manner that prevents it from participating in the association reaction. The relationship of these results to general pathways of protein precipitation is discussed.