Glutaraldehyde, the biological fixative of choice in the cytochemical localization of the phosphatases, was investigated for its effects on Pseudomonas aeruginosa alkaline phosphatase. Comparative studies on the inactivation of alkaline phosphatase by glutaraldehyde showed significant differences when the purified protein was compared with whole, cell-bound enzyme. The effects of the reagent on the kinetics of the purified enzyme were studied and some conclusions drawn as to the mode of inactivation. The reaction of glutaraldehyde with the cell envelope of P. aeruginosa was also investigated, and it was found not to modify the extraction of lipopolysaccharides from the outer membrane. This study emphasizes the care that must be taken to interpret data, cytochemical or otherwise, obtained when glutaraldehyde is used as a fixative or cross-linking reagent.