1. Upon controlled protein cleavage the catalytic activity of the Ca2(+)-ATPase from sarcoplasmic reticulum is drastically reduced concomitantly with small but significant changes in secondary structure as seen by Fourier transformed infrared (FT-i.r.) spectroscopy, although no loss of protein bound to the membrane is found. 2. FT-i.r. band fitting procedures show a reduction in the beta-sheet and turns content of the protein which is accompanied by an increase in alpha-helix and/or random structure. 3. These changes in the secondary structure of the protein appear to be well correlated to the tryptic digestion pattern and also to changes in the ATP hydrolysis rate of the Ca2(+)-ATPase. 4. It is concluded that these small changes reflect the disruption of key domains of the protein, which lie outside of the membrane matrix, leading to loss of enzymatic activity. 5. FT-i.r. spectroscopy appears to be a very useful technique to study changes in secondary structure of proteins.