Carbonylhemoglobin’s (HbCO’s) rapidly interconverting CO conformer bands are used as an indicator for the "limits of cryofixation". FTIR spectra of the CO stretching band region of HbCO in glassy aqueous solution obtained by hyperquenching at cooling rates of similar to 10(6)-10(7) K s(-1) and recorded at 78 K are compared with those of slow-cooled solutions, with either water or 75% (v/v) glycerol/water as solvent, which were recorded at several selected temperatures. The temperature dependence of the CIV/CIII conformer area ratios of HbCO in slow-cooled 75% glycerol/water solution is similar to that reported for the corresponding A(0)/A(1) ratios in carbonylmyoglobin. For HbCO in slow-cooled aqueous solution, a further increase of the CIV/CIII area ratios in freeze-concentrated solution is attributed to dehydration of HbCO. For hyperquenched HbCO solution the percent relative band area of the curve-fitted component band due to conformer CIV is between the values of the slow-cooled aqueous solution recorded at 293 and 270 K, and according to this criterion, the conformer CIV population is "frozen-in" in this temperature range. The estimate of HbCO’s conformer interconversion rate of >10(4) s(-1), or mean lifetime of <10(-4) a (Potter et al. Biochemistry 1990, 29, 6283-6295), gives an approximate value for the range of dynamic conformer equilibria which can be immobolized, or "frozen-in", close to ambient temperature by hyperquenching. It follows that the native state of biomolecules and its conformer population is better preserved in hyperquenched than in slow-cooled solution.