Soybean meal hydrolyzate (SMH) has been proposed for use as a natural preemergence herbicide. However, the mechanismby which SMH exerts its herbicidal activity remains unclear. In this paper, the herbicidal activities of SMH against perennial ryegrass (Lolium perenne L.) under non-sterile and sterile conditions were evaluated and the relationship between the molecular weight of the ultrafiltration (UF) fractions of SMH and their herbicidal activities were investigated.Besides, the ammonia content changes of the media of SMH treatments 7 d after incubation were also analyzed. The results showed that SMH inhibited the radicle growth of germinating L. perenne seeds in a dose-dependent manner under non-sterile condition. However, SMH in the concentrations investigated increased the radicle length and shoot length by 14-17% and 11-15% respectively under sterile condition. The ammonia contents in the SMH media at all treatments increased greatly from less than 0.01 mg L-1 to up to 11.86-41.37 mg L-1 after 7 d incubation under non-sterile condition.However, ammonia content did not change under sterile condition, proposing that the herbicidal activity might be caused by the free ammonia released from SMH by microbial activity. There was no difference on the perennial ryegrass radicle inhibition among the UF fractions of SMH on an equivalent N basis. It could be concluded that SMH exerted its herbicidal activity through the free ammonia released under non-sterile condition instead of by specific peptide(s) in SMH.