SUMMARYGlucose embedding is a simple and highly effective method for preparing biological macromolecules for high‐resolution electron microscopy. The investigation of conditions that can trap the M‐state intermediate in the bacteriorhodopsin (bR) photocycle has revealed, however, that when glucose‐embedded bR is prepared at ambient humidity, it does not fully retain the capability to execute a proper photocycle. However, ‘native’ photocycle properties are returned after glucose‐embedded samples are equilibrated at 81% relative humidity. Equilibration at relative humidities significantly higher than 81% causes glucose to dissolve in its own water of hydration, resulting in samples that may be too thick to be suitable for electron microscopy. The results obtained with bR indicate that caution should be taken with other biological specimens, and it cannot be assumed that glucose‐embedded biological macromolecules retain completely their native, hydrated structure, even when high‐resolution electron diffraction patterns are obtained. Equilibration of such samples at high humidity may generally be a worthwhile precaution when using the glucose‐em