The carotid body plays a crucial role in cardiorespiratory regulation. In the present study we investigated the effect of osmotic changes on cytoplasmic calcium concentration ([Ca2+]c) and pH (pHi) of isolated chemoreceptor cells of the rat carotid body. In CO2/HCO3−‐buffered medium, reduction of osmolality from the control level of 300 mosmol kg−1 to 250–285 mosmol kg−1 resulted in a rise in [Ca2+]c, as measured with Indo‐1, whereas elevation of osmolality to 350 mosmol kg−1 had no effect. The Ca2+ response required extracellular Ca2+ and was reduced by application of the L‐type Ca2+ channel antagonist nifedipine (10 μm). The hyposmosis‐induced Ca2+ response could be prevented by application of niflumic acid (300 μm), an inhibitor of the swelling‐activated Cl− channel. In whole‐cell patch‐clamp experiments niflumic acid abolished the swelling‐activated Cl− current but only slightly depressed the Ca2+ current. The inhibition of Ca2+ current by niflumic acid does not account for its action in preventing of hyposmosis‐induced Ca2+ response, which seems to be initiated by Cl−‐mediated depolarisation. Withdrawal of CO2/HCO3− also prevented the Ca2+ response. Reduction of the osmotic concentration by 50 mosmol kg−1 induced a small but sustained decrease in pHi, while elevation by 50 mosmol kg−1 had an inverse effect, as measured fluorimetrically with carboxy SNARF‐1. Our conclusion is that in the rat chemoreceptor cell the activation of Cl− channels, e.g. by hyposmotic challenge, induces depolarisation, which, in turn, activates voltage‐gated Ca2+ channels.