Genomic DNA profiles such as denaturing gradient gel electrophoresis (DGGE), temporal temperature gradient gel electrophoresis (TTGE), and single-strand conformation polymorphism (SSCP) have been commonly used to characterize bacterial communities in environmental samples. We recently developed a two-dimensional gel electrophoresis (2-DGE) method with a combination of chain-length polymorphism analysis (CLPA) and DGGE analysis, in order to improve the DNA resolution and resolve complex environmental DNA fragments produced by polymerase chain reaction (PCR) amplification. The 2-DGE method can generate high-resolution DNA separation maps on the basis of the lengths and composition polymorphisms of DNA sequences. It can thus facilitate detailed analyses between bacterial communities in complex environmental systems such as soil or water. For the present paper, we further developed two novel 2-DGE methods using a combination of CLPA and TTGE (or CLPA and SSCP) and here describe their potential application to the characterization of bacterial communities in nature using clustering analyses. The results show that DNA amplicons can undergo more detailed separation by the two new mapping than by their corresponding 1-DGE fingerprints. Our findings also suggest that these two new 2-DGE mapping techniques are more easily carried out than previously described DGGE-based 2-DGE mapping because they do not require a chemical denaturing gradient gel. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.