Recent studies have shown that integrons play a major role in spreading antibiotic resistance genes in clinical settings, as antibiotic resistance genes are frequently found located at gene cassettes. Polymerase chain reaction (PCR) analyses were carried out for the detection of the integrase genes of the three classes of integrons, and their gene cassettes were characterized in 118 clinical strains, including both gram-positive and gram-negative bacteria, isolated from a hospital in Guangzhou, China during 2004. Class 1 and 2 integrons were detected in 76.3% (90/118) and 0.8% (1/118) of the tested clinical isolates, respectively. Moreover four isolates were positive for both the integrons and no class 3 was detected. Some of them were first reported, such as Enterococcus faecalis, Enterococcus faecium, Alcaligenes sp., and Flavobacterium sp. Seven different arrays of gene cassettes of class 1 integron were found, and a high prevalence of dfrA12-orfF-aadA2 genes was observed. In addition, we identified a new none-open reading frame (ORF) cassette in a class 1 integron positive clinical coagulase-negative Staphylococcus strain GH69. The none-ORF cassette, a 700 bp sequence containing a 441 bp ORF and a 59-base element, is first characterized in this study. All class 2 integrons carried an array of gene cassettes dfrA1-sat2-aadA1. Moreover bacteria contain more than one integrons were also found in this study. Resistance to trimethoprim-sulfamethoxazole was frequently found to be encoded within integrons. Therefore, it is important that guidelines for the prudent use of antimicrobial agents are adopted and surveillance programs are established.