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To better understand the structure of macronuclear chromosomes in ciliates, the organization of macronuclear DNA was investigated in the hypotrich Kahliella matisi. Total DNA of K. matisi separated by agarose gel electrophoresis showed continuous smear ranging in size from ∼500bp to ∼15kb. This fragmentation was found to be due to the presence of gene-sized macronuclear chromosomes. The sequence analysis of four randomly cloned macronuclear chromosomes showed that K. matisi telomeres consist of 5'-dC4A4-3' repeats and carry one or two open reading frames. The transcription unit was found to be flanked with non-coding AT rich 5' leader and 3' trailer. No consensus transcription-regulatory sequences were identified in 5' leader and only one of analyzed gene-sized chromosomes showed the presence of conserved poly(A) addition signal sequence in 3' trailer. All ORFs showed highest relatedness to Oxytricha trifallax macronuclear chromosomes with conserved exon/intron structure. Sequence comparisons indicate that macronuclear chromosome organization is at least partially conserved in ciliates.