iAssembler is a standalone package to assemble ESTs generated using Sanger and/or Roche-454 pyrosequencing technologies into contigs. The pipeline gives much higher accuracy in EST assembly than other existing assemblers by employing an iterative assembly strategy and automated error corrections of mis-assemblies. iAssembler first performs iterative assemblies using MIRA and CAP3 (default: four cycles of MIRA assemblies followed by one CAP3 assembly) to correct assembly errors (mostly sequences derived from the same transcript fail to be assembled together) which occur frequently in just one round of assembly. The program then performs post-assembly quality checking by 1) aligning each EST sequence to its corresponding unigene sequence to identify mis-assemblies; and 2) performing all-verus-all pair-wise sequence alignments of unigenes to identify sequences derived from same transcripts that fail to be assembled together. The identified mis-assemblies are then corrected by the program automatically.