The initial event in the regulatory volume increase (RVI) of rat hepatocytes is an influx of Na+ that is then exchanged for K+ via stimulation of Na+/K+-adenosine triphosphatase (ATPase). In this study, we analysed the activation pattern of the Na+ transporters underlying RVI as a function of the degree of hypertonic stress. In confluent primary cultures, four hypertonic conditions were tested (changes from 300 to 327, 360, 400 or 450 mosmol/l) and the activities of Na+ conductance, Na+/H+ antiport, Na+-K+-2Cl- symport and Na+/K+-ATPase were quantified using intracellular microelectrodes, microfluorometry and time-dependent, furosemide- or ouabain-sensitive 86Rb+ uptake, respectively. Neither Na+ conductance nor Na+-K+-2Cl- symport responded to 327 mosmol/A. At 360, 400 and 450 mosmol/l, uptake via these transporters would lead to increases of cell Na+ by 33.0, 49.0 and 49.0 and by 4.5, 10.4 and 9.2 mmol/l per 10 min, respectively. In contrast, Na+/H+ antiport exhibited 65% of its maximal activation already at 327 mosmol/l. At the four osmolarities tested, this transporter would augment cell Na+ by 6.9, 8.9, 9.8 and 10.6 mmol/l per 10 min. The sums of Na+ import were consistent with the amounts of Na+ exported via Na+/K+-ATPase plus the actual increases of cell Na+ (21.2, 58.5, 63.6 and 68.3 mmol/l per 10 min and 2.2, 4.0, 6.3 and 8.2 mmol/l, respectively). In addition, these elevations of cell Na+ plus the increases of cell K+ (via Na+/K+-ATPase) that amounted to 5.0, 6.5, 17.5 and 18.4 mmol/l were consistent with the increases of intracellular osmotic (cationic) activity of 2.5, 11.5, 21.0 and 28.5 mmol/l, respectively, computed from RVI data. It is concluded that the principle of rat hepatocyte RVI, i.e. an initial uptake of Na+ that is then exchanged for K+ via Na+/K+-ATPase, is realized over the entire range of 9-50% hypertonicity tested. The set-point for the activation of RVI clearly lies below 327 mosmol/l. Na+/H+ antiport is the most sensitive Na+ importer involved in RVI, whereas Na+ conductance plays the prominent role from 360 mosmol/l upwards.