Background Electrolyte and acid-base balance may be differently affected by the infusion mode in on-line hemodiafiltration (HDF). We studied the effects of the different infusion modes on bicarbonate transport across the dialyzer membrane, and thus on the final bicarbonate balance of the HDF sessions. Methods Instantaneous HCO3− transfer across the dialyzer membrane, blood bicarbonate profile and the total balance of the sessions were studied in six dialysis patients under the same operating conditions over 36 HDF sessions, in order to compare the effects of predilution HDF (pre-HDF), postdilution HDF (post-HDF), and mixed HDF on the final bicarbonate balance. Results The final HCO3− balance was more positive in post-HDF vs pre-HDF (142 ± 36 vs 99 ± 41 mmol/session, p<0.05), with a final blood HCO3− concentration of 26.6 ± 1.0 vs 25.4 ± 1.1 mmol/L, (p<0.05). Mixed HDF yielded intermediate results (balance: 119 ± 42 mmol/session, final HCO3− 26.2 (1.2 mmol/L). These differences were seen to result from the increased HCO3- concentration of blood entering the filter in predilution, due to the infused HCO3−, enhancing convective loss and reducing the driving force for diffusive HCO3− gain. Conclusions Bicarbonate concentration in dialysate-reinfusate is critical in order to obtain an adequate end of session HCO3− balance in on-line HDF. The predilution method produced the lowest cumulative net HCO3− gain between the three studied infusion modes. Our data suggest that, under the same operating conditions and excluding the effect of ultrafiltration, dialysate HCO3− should be increased by about 2 mmol/L in pre-HDF, and 1 mmol/L in mixed HDF, to yield the same final balance as in post-HDF.