The present study investigated the mechanical elongation of the centromere in the barley chromosomes by a microneedle manipulation method for the structural analysis of the chromosomes. Chromosomes were extracted from barley root cells, affixed on a cover slip by a standard preparation method, and elongated in either distilled water, phosphate buffered saline (PBS), or 2 x sodium saline citrate (SSC). The mechanical property of the chromosome elongation was assessed by the measurement of the force required for the elongation of chromosomes. This assessment has shown that the chromosomes in distilled water were much firmer than those in the PBS or 2 x SSC. To confirm the elongation of the centromere, the elongated chromosomes were investigated by fluorescence in situ hybridization with a centromere probe. The fluorescence information indicated that the extent of the loosening of the centromere during elongation differed depending on the buffers used; the centromere elongated in 2 x SSC was more loosened than that in the PBS. Atomic force microscopy also revealed the structure of the unpacked centromere after the mechanical elongation, when rows of fibrous structures about 30 to 50 nm thick were clearly observed in the centromere elongated in 2 x SSC. The investigation of elongated chromosomes should prove useful for an understanding of the structural analysis of chromosomes.