To investigate the fate of penicillin G (PEN G) in the wastewater from a PEN G production facility and the receiving river, an analytical method was developed for the simultaneous detection of PEN G and five degradation products using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS). PEN G had already undergone transformation before entering into the wastewater treatment plant (WWTP), with concentrations of 153+/-4 microg/L in raw wastewater. Most of the PEN G could be eliminated following successive treatments of anaerobic, hydrolysis, and two aerobic units under a hydraulic residence time (HRT) of 30 h, and the final concentrations were 1.68+/-0.48 microg/L in treated water. In the receiving river, the concentration of PEN G decreased from 0.31+/-0.04 microg/L at the discharging point to under the detection limit (0.03 microg/L) at the last sampling site (about 30 km from the discharging point). The main PEN G degradation products in surface water were found to be penilloic acid, penicilloic acid and isopenillic acid, which occupied 65.8%, 20.4% and 12.9%, respectively, of the total concentration at the last site. This is the first study on the behaviors of PEN G and its main degradation products in wastewater treatment processes and the aquatic environment.