Elevated osmolarity is known to inhibit secretion from a wide range of cells including bovine adrenal chromaffin cells. The mechanism of this inhibition is unclear but the elevated osmolarity has been proposed to oppose an osmotic driving force involved in exocytotic fusion. Using the fluorescent indicators quene 1 and fura2, we monitored the effect of elevated osmolarity on cytoplasmic pH (pHi) and cytoplasmic free Ca2+ [( Ca2+]i). Elevated osmolarity increased both pHi and [Ca2+]i, but had no effect on the [Ca2+]i rise elicited by either K+ or nicotine. Elevating pHi with NH4Cl was shown to inhibit secretion from chromaffin cells. The elevation of pHi by hyperosmolar solutions is proposed as one of the mechanisms by which elevated osmolarity inhibits secretion.