Changes in the secondary structure and aggregation of chymotrypsinogen were investigated by infrared difference spectroscopy in conjunction with temperature and pressure tuning IR spectroscopy; both the amide I' band and side chain bands were studied. A prominent component of the amide I' band in the difference spectrum obtained upon cooling a chymotrypsinogen solution, or increasing the hydrostatic pressure, was observed in the region between 1627 and 1622 cm-1. Under denaturing conditions a white gel was formed, which is attributed to irreversible self-association or aggregation. This process was accompanied by the appearance of two new amide I' bands in the infrared spectrum of the protein: a very strong band at 1618 cm-1 and a weak band at 1685 cm-1. These bands are assigned to peptide segments with anti-parallel aligned beta-strands.