This is the first report describing the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as a protein associated with the cell envelope of a gram-negative bacterium (Aeromonas hydrophila). Dose-dependent GAPDH activity was detected in whole bacterial cells from exponentially growing cultures, indicating that an active form of GAPDH is located outside the plasma membrane. This activity represents roughly 10-20% of total cell activity, and it is not reduced by pretreatment of the cells with trypsin. Assays with soluble GAPDH indicate that the activity measured in intact cells does not originate by rebinding to intact cells of cytosolic enzyme released following cell lysis. GAPDH activity levels detected in intact cells varied during the growth phase. The relationship between GAPDH activity and cell culture density was not linear, showing this activity as a major peak in the late-logarithmic phase (A600 = 1.1-1.3), and a decrease when cells entered the stationary phase. The late exponential growing cells showed a GAPDH activity 3 to 4-fold higher than early growing or stationary cells. No activity was detected in culture supernatants. Enzymatic and Western-immunoblotting analysis of subcellular fractions (cytosol, whole and outer membranes, and periplasm) showed that GAPDH is located in the cytosol, as expected, and also in the periplasm. These results place the periplasmic GAPDH of A. hydrophila into the family of multifunctional microbial cell wall-associated GAPDHs which retain their catalytic activity.