The pH regulation of rainbow trout and carp thrombocytes was investigated using the pH-sensitive fluorescent dye BCECF and a PTI Imagescan image analysis system. The cells were experimentally acidified either using the ammonium chloride prepulse method or perfusing the cells with a saline containing propionate ion. The recovery from the acidification was followed. By using ion substitutions (N-methyl-D-glucamine for sodium; TRIS vs carbon dioxide/bicarbonate buffer) and transport inhibitors (amiloride to inhibit sodium/proton exchange and DIDS to inhibit chloride and bicarbonate dependent acid extrusion mechanisms) the transport pathways were identified. The pH regulatory mechanisms of thrombocytes in both species were similar. The pH recovery after experimental acidification was fully inhibited by removing sodium from the medium. The sodium-dependent acid extrusion had both amiloride-sensitive and DIDS-sensitive components, indicating that both sodium/proton exchange and sodium-dependent bicarbonate transport were involved. The role of sodium-dependent bicarbonate movements in pH regulation was ascertained by showing that pH recovery occurred faster at a high than at a low bicarbonate concentration. Adrenergic stimulation did not affect the intracellular pH.