Using the weak base 3-(2,4-dinitroanilino)-3'-amino-N-methyI propylamine (DAMP) in in situ probing experiments, ultrastructural evidence for the acidic nature of the thylakoids in whole cells has been obtained in three unicellular pennate diatom species, with special emphasis on Navicula salinarum. Positive internal controls were provided by compartments known to be acidic (microbodies, lysosomal active vacuoles, and silica deposition vesicle). Negative internal controls were circumneutral compartments (nucleus and mitochondria). The predictable labelling of these compartments demonstrated the translocation of unprotonated DAMP over membranes and into acidic environments, where it became protonated. In cells that were treated with ionophores to disrupt pH gradients after incubation with DAMP, DAMP was no longer confined specifically to thylakoids and appeared to be more randomly distributed within the chloroplasts, indicating that the pH gradient of the thylakoids was affected. After densitometry, the transthylakoid DeltapH was estimated to vary between 1.0 and 2.0, coinciding with a moderate lumen acidity. The results obtained provide what we believe is the first clear evidence of the acidic nature of the thylakoid lumen in living cells at an ultrastructural level.