The effect of pH, monovalent cations, glycerol, temperature, and pressure on the carbonmonoxy (CO) stretching mode of camphor-bound cytochrome P-450cam (CYP 101) was studied. Two effects, band overlap and frequency shift, have been observed. The CO stretch infrared band located at about 1940 cm-1 is asymmetric because of the overlap of three bands at about 1931 cm-1, 1939 cm-1, and 1942 cm-1 with strongly different populations. Reducing the temperature or increasing the pressure leads to splitting the band or switching the asymmetry from the lower energy side to the higher energy side of the infrared band. The overlap of several CO stretch bands indicates conformational substates within the heme pocket. A frequency shift of the predominantly populated band is observed by changing all the parameters mentioned. The pH-induced frequency shift follows an S-shape with the pK at 6.2, which matches the pK observed for the pH-induced high-spin/low-spin transition. Conformational changes on the proximal heme side are suggested to be the origin. Monovalent cations at saturating concentration induce a small frequency shift depending on the ion radius. The potassium ion is the one that induces a CO stretch frequency with the highest wave-number while sodium and lithium (smaller radii) and rubidium and caesium ion (larger radii) have diminished values, which is supporting evidence for the special function of the potassium ion within the structure. Glycerol and hydrostatic pressure induce a red shift of the CO stretching frequency. Forced contact of the polar hydroxyl group of Thr252 of the I helix induced by pressure and indirectly by glycerol is suggested to change the CO dipole moment, reflecting in the decreased CO stretching frequency.