A sensitive method for simultaneous determination of azithromycin (AZI), acetylspiramycin (ACE), erythromycin (ERY), and josamycin (JOS) was developed by CE coupled with electrochemilurninescence detection with Ru(bpy)(3)(2+). The parameters related to separation and detection were investigated in detail. The four macrolides were well separated and detected within 6 min under the optimized conditions. The LOD (S/N = 3) of AZI, ACE, ERY, and JOS were 1.2 x 10(-9), 7.1 x 10(-9), 3.9 x 10(-8) and 9.5 X 10(-8) mol/L, respectively. The LOQ (S/N= 10) of AZI, ACE, ERY, and JOS in human urine were 8.2 x 10(-8), 2.5 x 10(-7), 8.9 x 10(-7) and 1.2 x 10(-6) mol/L, respectively. The recoveries of the four macrolides in human urine and pharmaceutical tablet samples were 85.0-104.0% at different concentration levels.