The renewable silver-amalgam film electrode (Hg(Ag)FE) was applied for voltammetric characterization and determination of semi-synthetic macrolide antibiotics azithromycin (AZI), clarithromycin (CLA) and roxithromycin (ROX) in the Britton-Robinson buffer as supporting electrolyte ranging the pH from 4.0 to 11.9. All three macrolides showed reduction signals in fairly negative potential range. During direct cathodic square wave voltammetric (SWV) investigations conducted over the potential range from -0.75 V to -2.00V vs SCE, either one or two reduction peaks were obtained in the potential range from -1.5 to -1.9 V. The shapes and intensities of the signals depend on the applied pH values in wider pH ranges. For analytical purposes concerning the development of direct cathodic SWV and adsorptive stripping SWV (SW-AdSV) methods the neutral and slightly alkaline media were suitable as pH 7.2, pH 7.4 and pH 7.0 for AZI, CIA and ROX, respectively. Based on the cyclic voltammograms recorded at these pH values, adsorption-controlled electrode kinetics process can be proposed for all three macrolides. Furthermore, the water suppressed H-1 NMR measurements in the pH range between 6.0 and 10.5 indicated that the macrolide molecules at the optimal analytical conditions are predominantly in protonated form via their tertiary amino groups which supported in all three cases their adsorption on the appropriately polarized Hg(Ag)FE electrode. The optimized direct cathodic SWV methods showed good linearity in concentration ranges 4.81-23.3 mu gmL(-1), 1.96-28.6 mu gmL(-1) and 1.48-25.9 mu gmL(-1) for AZI, CIA and ROX, respectively. The development of the SW-AdSV methods resulted in the linear responses at lower concentration ranges as 1.0-2.46 mu gmL(-1), 0.05-0.99 mu gmL(-1) and 0.10-0.99 mu gmL(-1), for AZI, CIA and ROX, respectively. The relative standard deviation for all developed methods was not higher than 1.0% except the SWV method for AZI with 4.7%. In the case of all three investigated macrolide antibiotics the protonated form of the tertiary amino group(s) at appropriate accumulation potential and time favored the adsorption of the ionic form of the target molecules offering the opportunity for the development of SW-AdSV methods for their trace level analysis on Hg(Ag)FE. Optimized SW-AdSV method was applied for determination of ROX in pharmaceutical preparation Runac. (C) 2017 Elsevier Ltd. All rights reserved.