The chemosensitive response of locus coeruleus (LC) neurones to changes in intracellular pH (pH(i)), extracellular pH (pH(o)) and molecular CO(2) were investigated using neonatal rat brainstem slices. A new technique was developed that involves the use of perforated patch recordings in combination with fluorescence imaging microscopy to simultaneously measure pH(i) and membrane potential (V(m)). Hypercapnic acidosis (15 % CO(2), pH(o) 6.8) resulted in a maintained fall in pH(i) of 0.31 pH units and a 93 % increase in the firing rate of LC neurones. On the other hand, isohydric hypercapnia (15 % CO(2), 77 mM HCO(3)(-), pH(o) 7.45) resulted in a smaller and transient fall in pH(i) of about 0.17 pH units and an increase in firing rate of 76 %. Acidified Hepes (N-2-hydroxyethylpiperazine-N'-2- ethanesulfonic acid)-buffered medium (pH(o) 6.8) resulted in a progressive fall in pH(i) of over 0.43 pH units and an increase in firing rate of 126 %. Isosmotic addition of 50 mM propionate to the standard HCO(3)(-)-buffered medium (5 % CO(2), 26 mM HCO(3)(-), pH(o) 7.45) resulted in a transient fall in pH(i) of 0.18 pH units but little increase in firing rate. Isocapnic acidosis (5 % CO(2), 7 mM HCO(3)(-), pH(o) 6.8) resulted in a slow intracellular acidification to a maximum fall of about 0.26 pH units and a 72 % increase in firing rate. For all treatments, the changes in pH(i) preceded or occurred simultaneously with the changes in firing rate and were considerably slower than the changes in pH(o). In conclusion, an increased firing rate of LC neurones in response to acid challenges was best correlated with the magnitude and the rate of fall in pH(i), indicating that a decrease in pH(i) is a major part of the intracellular signalling pathway that transduces an acid challenge into an increased firing rate in LC neurones.