To test the hypothesis that silicification occurs under acid conditions in the silicon deposition vesicle (SDV), the acidity of the SDV of the pennate diatoms Navicula pelliculosa (Brébisson et Kützing) Hilse, N. salinarum (Grunow) Hustedt, and Nitzschia sigma (Kützing) Smith was determined during development of new frustule valves. Cells were incubated with the weak base 3-(2,4-dinitroanilino)-3′-amino-N-methylpropylamine (DAMP) followed by immunocytochemical localization in whole cells and on ultrathin sections. After resupplying silicate to cells synchronized by silicon depletion, the uptake of this nutrient from the medium was the same with or without DAMP; new valves developed without morphological aberrations that could conceivably have been caused by the probe. DAMP was found in cellular compartments known to be acidic, such as vacuoles active as lysosomes, the lumen of thylakoids, and microbodies. In the nucleus and mitochondria, which are circumneutral and basic compartments, the probe did not appear. Besides its presence in acidic compartments, DAMP was specifically accumulated within the SDV during formation of new valves; during the process of valve maturation, the SDV seemed to become increasingly acidic. In control experiments using the ionophores chloroquine, valinomycin, and nigericin, the compartmental location of DAMP was clearly disturbed, resulting in a random intracellular distribution. Accumulation of the fluorescent probe rhodamine 123, which can be translocated over membranes by a reducing potential, confirmed that the SDV can translocate weak bases. The results with DAMP suggest that the pH of the SDV is important in the silicification of diatoms: It facilitates a fast nucleation and aggregation of silica particles, thus increasing the rate of formation of the mature frustules. In addition, the acidic environment might protect the newly formed valves against dissolution before completion and coverage by the organic casing prior to their secretion.