The H+-ATPase rich (HR) cells of zebrafish larvae are a sub-type of ion-transporting cell located on the yolk sac epithelium that are responsible for Na+ uptake and H+ extrusion. Current models of HR cell ion transport mechanisms in zebrafish larvae are well established, but little is known about the involvement of the various ion transport pathways in regulating intracellular acid-base status. In the present study, a ratiometric imaging technique using the pH indicator dye BCECF was developed to monitor intracellular pH (pHi) continuously in larval zebrafish HR cells in vivo. Initial validation experiments demonstrated that HR cells subjected to respiratory acidosis (1% CO2) or metabolic alkalosis (20 mM NH4Cl) exhibited changes in BCECF 513/438 emission ratios which were consistent with the expected effects of these treatments on pHi. Subsequent experiments focussed on the involvement of the two principal apical membrane acid excretory pathways, the Na+/H+ exchanger (isoform NHE3b; zslc9a3.2) and the H+-ATPase (atpv1aa) in pHi regulation. Additionally, the role of HR cell carbonic anhydrase (“CA2-like a”) was investigated because of its presumed role in providing H+ for Na+/H+ exchange and H+-ATPase. To do so, relative HR cell pHi changes were monitored during acid-base challenges in shams and in fish experiencing morpholino gene knockdown of either NHE3b, H+-ATPase or “CA2-like a”. The temporal pattern and extent of intracellular acidification during exposure of fish to 1% CO2 and the extent of post-CO2 alkalization were altered markedly in fish experiencing knockdown of “CA2-like a”, NHE3b or H+-ATPase. Although there were slight differences among the three knockdown experiments, the typical response was a greater degree of intracellular acidification during CO2 exposure and a reduced capacity to restore pHi to baseline levels post-hypercapnia. Knockdown of “CA2-like a”, although presumed to limit H+ availability to NHE3b and H+-ATPase, yielded qualitatively similar results to knockdown of either single H+ excretory pathway. The metabolic alkalosis and subsequent acidification associated with NH4Cl exposure and its washout were largely unaffected by gene knockdown. Overall, the results suggest markedly different mechanisms of intracellular acid-base regulation in zebrafish HR cells depending on the nature of the acid-base disturbance.