The formation of native disulfide bonds during in vitro protein folding can be limiting in obtaining biologically active proteins. Thus, optimization of redox conditions can be critical in maximizing the yield of renatured, recombinant proteins. We have employed a folding model, that of the p subunit of human chorionic gonadotropin (hCG-p), to investigate in vitro oxidation conditions that facilitate the folding of this protein, and have compared the in vitro rates obtained with the rate of folding that has been observed in intact cells. Two steps in the folding pathway of hCG-p were investigated: the rate-limiting event in the folding of this protein, and the assembly of hCG-p with hCG-a. The rates of these folding events were determined with and without protein disulfide isomerase (PDI) using two different types of redox reagents: cysteamine and its oxidized equivalent, cystamine, and reduced and oxidized glutathione. Rates of the rate-limiting folding event were twofold faster in cysteamine/cystamine redox buffers than in glutathione buffers in the absence of PDI. Optimal conditions for hCG-p folding were attained in a 2 m M glutathione buffer, pH 7.4, that contained 1 mg/mL PDI and in 10 m M cysteamine/cystamine, pH 8.7, without PDI. Under these conditions, the half-time of the ratelimiting folding event was 16 to 20 min and approached the rate observed in intact cells (4 to 5 min). Moreover, folding of the p subunit under these conditions yields a functional protein, based on its ability to assemble with the a subunit. The rates of assembly of hCG-p with hCG-a in the cysteamine/cystamine or glutathione/PDI redox buffers were comparable (ti,* = 9 to 12 min). These studies show that rates of folding and assembly events that involve disulfide bond formation can be optimized by a simple buffer system composed of cysteamine and cystamine.