Acridine orange (AO) trapping in conjunction with fluorescence microscopy was applied toPammecium cells. Trichocysts were not labeled when analyzed with an image intensification system (as opposed to a lysosomal population). Only with increasing intensity of ultraviolet light (UV) did trichocysts (and to some extent the cytosol) exhibit orange fluorescence, both effects being paralleled by inaeasing cell damage. Therefore, in comparison with the reported cytosolic pH (6.8), trichocysts cannot be considered as essentially acidic compartments. This is supported by experiments in vitro, using isolated cortex fragments or isolated fractions of membrane-bounded trichocysts (390% non-leaky). Again, during w illumination orange fluorescence was observed even in the absence of ATP and Mg2'. Furthermore, this A0 fluorescence and the condensation state of trichocyst contents were not affected by N H 3 or by any of the widely differing ion-and H+-exchange inhibitors or ionophores tested. Decondensation of trichocyst contents occurred only when Ca2+ ionophore A23187 or X537A was incorporated into trichocyst membranes and when Ca2+ was then added. In this case all trichocysts partially decondensed within their intact membranes. We conclude that A 0 might be trapped in trichocysts by the abundant acidic secretory components during observation with uv light, rather than by acidic luminal pH. ( J Hisrochem Cyrochem 40953-160, 1992)