Mutational analysis was performed to probe the development of hydrophobic clusters during the early events in the folding of dihydrofolate reductase. Replacements were made in several hydrophobic subdomains to examine the roles of hydrophobicity and stereochemistry in the formation of kinetic intermediates. Amide protons in two of these clusters, including residues I91, I94, and I155, have been shown to be protected against solvent exchange within 13 ms of folding. Additional hydrophobic clusters were probed by substitutions at residues I2, I61, and L112; these residues are not protected from exchange until later in the folding reaction. Valine and leucine replacements at positions I91, I94, and I155 significantly diminish the formation of the burst phase kinetic intermediate, relative to the wild-type protein. In contrast, I2 and I61 are insensitive to these substitutions in the first 5 ms of the folding reaction, as is the replacement of L112 with either isoleucine or valine. These results demonstrate that the tightly packed core of dihydrofolate reductase is acquired in a non-uniform fashion, beginning in the submillisecond time frame. The progressive development of specific side-chain packing in localized hydrophobic clusters may be a common theme for complex protein folding reactions.