SUMMARY1. We have investigated the use of TMA-DPH (1-[4-(trimethylammonio)phenyl]-6-phenylhexa-1,3,5-triene) as an indicator of exocytosis in individual bovine anterior pituitary cells using microfluorimetric imaging.2. TMA-DPH was photolabile in artificial and cell membranes. In cells incubated in TMA-DPH the distribution of fluorescence depended both on the incubation time and the illumination schedule. If the dye was added while the cells were subjected to repeated cycles of 0-36 s light intermittent with 1-15 s dark, the fluorescence of the peripheral annulus and the central region of individual cells rose in parallel and reached a steady state within 200 s; the annulus was always brighter than the central region. However, using long intervening dark periods (200 s), the central region continued to incorporate dye after the annulus had reached a plateau.3. When the cells were loaded with TMA-DPH using intermittent light with short dark periods, the dye washed out of the central region and the annulus in parallel when external dye was removed. However, if the cells had been loaded using long dark periods, the dye was washed out of the central region more slowly than from the annulus.4. When cells were incubated in TMA-DPH in the dark for 1 min and then exposed to constant illumination in the presence of external dye, the fluorescence of the central region and the annulus both decayed in parallel to a new steady state. If the cells were incubated in TMA-DPH in the dark for 240 min the fluorescence from each region fell to a steady state but the falls were larger and were not in parallel.5. We suggest that TMA-DPH fluorescence was derived from plasma membraneassociated and internalized dye and that the amount of fluorescence from the latter varied because TMA-DPH was photobleached. Thus, when illumination was interrupted by short dark intervals, annular fluorescence was high compared to central fluorescence because bleached dye in the plasma membrane was rapidly replaced by unbleached dye from the medium. However, long dark intervals permitted the dye to be internalized before it was bleached and fluorescence was therefore also present in central regions.* To whom reprint requests should be addressed.